SF 

187 

.D3 



Issue(> June 12, 1911. 



\ 



U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 137. 

A. D. MELVIN, Chief OF Bureau. 



ANTHRAX, 



WITH SPECIAL REFERENCE TO THE PRODUCTION 

OF IMMUNITY. 



BY 



CHARLES F. DAWSON, M. D., D. V. S., 
Veterinarian, Delaware College Agricultural Experiment Station^ 

UNDER THE DIRECTION OF 

JOHN R. MOHLER, Vi M. D., 

Chief of the Pathological Division y 
Bureau of A nimal Industry . 




Q 



WASHINGTON: 
GOVERNMENT PRINTING OFFICE. 

■ 1911. 



II 




Qass ^T^l 

Book ' ' , . 



n 



Issued June 12, 1911. 

U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 137. 

A. D. MELVIN, Chibf of Bureau. 



ANTHRAX, 



WITH SPECIAL REFERENCE TO THE PRODUCTION 

OF IMMUNITY. 



, BY 

CHARLES F. DAWSON, M. D., D. V. S., 
• * 

Veterinarian, Delaware College Agricultural Experiment Station^ 

UNDER THE DIRECTION OF 

JOHN R. MOHLER, V. M. D., 

Chief of the Pathological Division^ 
Bureau of Animal Industry. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1911. 



t^^ 






THE BUREAU OF ANIMAL INDUSTRY. 



Chief: A. D. Melvin. 
Assistant Chief: A. M. Farrington. 
Chief Clerk: Charles C. Carroll. 

Animal Husbandry Division: George M. Rommel, chief. 
Biochemic Division: M. Dorset, chief. 
Dairy Division: B. H. Rawl, chief. 

Inspection Division: Rice P. Steddom, chief; Morris Wooden, R. A. Ramsay, 
and Albert E. Behnke, associate chiefs. 
Pathological Division: John R. Mohler, chief. 
Quarantine Division: Richard W. Hickman, chief. 
Zoological Division: B. H. Ransom, chief. 
Experiment Division: E. C. Schroeder, superintendent. 
Editor: James M. Pickens. 



lil 



I 



9 

1 



LETTER OF TRANSMITTAL 



U. S. Department ®f Agriculture, 

BuiiEAu OF Animal Industry. 

Washington, D. C, March W, 1911. 

Sir : I have the honor to transmit for publication the accompan^ang 
manuscript on the subject of anthrax, by Dr. Charles F. Dawson, of 
the Delaware College AgTicultural Experiment Station, the experi- 
mental work having been carried out in cooperation with the Patho- 
logical Division of this bureau. 

Anthrax is one of the oldest and most widely distributed diseases of 
animals, and is particularly destructive to cattle and sheep. The 
existing method of combating the infection is to endeavor to control 
it by means of a vaccine devised by Pasteur. Two injections are, 
however, necessary by this method, and an active immunity is not 
established for about four weeks. 

Two important results have been accomplished through the work 
described in this paper. A single vaccine has been produced which 
may be substituted for the above-mentioned double vaccine, thus cut- 
ting down the time one-half. The other result is the production of 
an antianthrax serum that will confer an immediate passive immu- 
liitj. Thus the two combined are a distinct advantage over the exist- 
ing method in the case of sudden outbreaks, where there is no time to 
prepare for the disease in the usual wa}^. This serum can also be used 
in conjunction with the double vaccine of Pasteur. 

I recommend that the paper be published in the bulletin series of 
this bureau. 

EespectfuUy, A. D. ^Jelvin, 

Chief of Bwreaii. 

Plon. James Wilson, 

Secretary of Agriculture. 

8 



CONTENTS. 



Page. 

Introduction 5 

Historical 5 

The anthrax bacillus 6 

Cultural and morphological characters < 7 

Channels of infection 9 

Infection of the soil 10 

Methods of diagnosis 11 

Preparation of specimens , 11 

Animal inoculations 12 

Different forms of anthrax 13 

Symptoms. 14 

Post-mortem appearances 15 

The cause of death 15 

Disposal of carcasses Ig 

The official method in Delaware 17 

Susceptibility of the various animals 17 

Anthrax in man 19 

Malignant pustule 19 

Pulmonary and intestinal anthrax 20 

The anthrax season 20 

Prevalence of anthrax in Delaware 22 

Methods of producing immunity in animals 24 

The existing methods of double vaccination (Pasteur method) 24 

Effectiveness of the method 25 

A test of Pasteur vaccines 26 

Experiments with various substances to test immunizing powers 28 

Anthraxin 28 

Pyocyanase 30 

Anthraxase 35 

Anthraxoin 38 

A commercial vaccine in pill form : 40 

Preparation of an effective single vaccine 41 

Tests of the single vaccine 42 

A serum for producing immediate immunity 43 

Experiments with the serum 44 

Conclusion 47 

4 



ANTHRAX, 

WITH SPECIAL REFERENCE TO THE PRODUCTION OF IMMUNITY. 



INTRODUCTIOlSr. 

Anthrax is primarily a disease of herbivorous animals, and occurs 
as an epizootic in sheep, cattle, horses, and mules, the animals being 
susceptible in the order named. In experimental work the small ani- 
mals, such as mice, guinea pigs, and rabbits, are extremely suscep- 
tible. The French name for the disease is " charbon ; " the German, 
'' Milzbrand." "WTien it occurs in domestic animals it is also some- 
times known as splenic fever. In natural outbreaks anthrax usually 
has the character of a rapidly fatal septicemia, with the presence of 
large numbers of the characteristic bacilli in the blood. 

WTiile the disease does not occur as a natural infection in man in 
the same sense as in animals, man frequently becomes infected from 
handling infected animals or their products, such as hides and hair. 
"When man becomes infected through abrasions of the skin, the result- 
ing disease process is carbunculous in nature and is known as " malig- 
nant pustule." "\Anien the infection takes place in the lungs, the dis- 
ease is known under the name " woolsorters' disease." A third form 
in man may attack the intestinal tract. The latter two forms are 
rapidly fatal. In malignant pustule the disease may remain localized 
and the patient may recover. If, however, the bacilli migrate from 
the local lesion to the blood stream, death rapidly ensues, the patient 
dying from septicemia or toxemia. 

HISTORICAL. 

Anthrax is one of the oldest diseases. Moses records it in Exodus 

9 : 9. Homer, Ovid, Plutarch, Yirgil, Pliny, and many others have 

mentioned it in their writing. It exists in all countries and in all 

latitudes. It was formerly very destructive to human life as well 

as to animals. In 1617, near Naples, 60,000 people are reported to 

have died from anthrax. In San Domingo, in 1770, 15,000 persons 

perished in the short period of six weeks. This enormous death rate 

was probably due, in part, to eating the carcasses of animals dead 

from the disease. 

5 



6 ANTHRAX. 

It was not until the middle of the nineteenth century that scientists 
began the serious study of this very destructive disease. In 1849, 
Poilender, while studying the blood of animals dead from anthrax, 
discovered numerous rod-shaped, microscopic bodies, which he 
claimed were the cause of the disease. This claim remained unsub- 
stantiated until 1863, when Davaine announced that the bodies dis- 
covered by Pollender were bacteria, and showed that the blood of an 
animal could not cause anthrax in another animal when injected into 
it unless it contained these bodies. To this bacterium Davaine gave 
the present name, Bacillus anthracis. Davaine^s views were not 
immediately accepted, but others took up the work, and in 1876 Dr. 
Kobert Koch, in making his first contribution to bacteriology, 
announced that he had been successful in proving the correctness of 
Davaine's work on anthrax, and the question was then accepted as 
settled. Koch isolated the bacilli in pure culture and demonstrated 
their ability to form spores. He claimed that natural infection takes 
place through the intestinal tract, although he was at first unable 
to demonstrate this by feeding food infected by artificial cultures. 
Later, Pasteur succeeded in producing anthrax by feeding hay 
sprayed with cultures of Bacillus anthracis when he at the same time 
mixed with the hay thistles or any plants that would cause pricking 
or abrasions of the intestinal tract so that the bacilli could pass into 
the tissues. 

THE ANTHSAX BACILLUS. 

On account of the comparatively large size of the Bacillus anthra- 
cis- — it being from 5 to 20 micromillimeters long and from 1 to 1.5 
micromillimeters broad, although both longer and shorter forms 
occur — and the great readine^ and certainty with which it kills 
experimental animals, it was the earliest disease to be studied with 
the microscope and hj modern bacteriological methods. We first 
learned to demonstrate spore formation and spore staining from 
anthrax cultures. The germ is exceedingly easy to cultivate, gTOw- 
ing on almost any medium and at a wide range of temperature. It 
has many of the general morphological characters of the whole 
group of bacteria, and is, therefore, very useful for teaching pur- 
poses. The bacillus has a place in the earliest researches on im- 
munity, Pasteur having shown in 1880-1882 that animals inoculated 
with strains of the bacilli which had been devitalized to a certain 
extent by cultivation at high temperatures did not contract anthrax 
when exposed, while those not so protected did contract the disease. 
Spores of anthrax are among the most resistant things in nature, 
and are, therefore, useful in testing the germicidal power of disin- 
fectants. 

If a drop of blood is taken immediately after death from an animal 
dead from anthrax and examined with a microscope, large numbers 



THE ANTHRAX BACILLUS. 7 

of nonmotile bacteria will be seen. A much better view of them 
can be obtained if a so-called blood-smear preparation is stained 
with methylene blue or any of the basic anilin dyes. It will be 
noticed that the ends of all the bacilli are sharply cut across. If 
viewed with a higher power, it will be seen that the ends bulge at 
the corners, making the ends appear slightly concave. The cells are 
also noticed to have a capsule, and when several cells are lying end 
to end the capsule seems common to the entire chain of cells. 

If in making preparations of anthrax blood great care is taken 
not to overheat, but just enough to " set " the specimen, and methy- 
lene blue is used as the stain, we can, while viewing the slide by 
reflected light with the unaided eye, notice a violet coloration of the 
entire specimen„ If we now place the slide under the microscope, 
we can see that the space between the bacillus and its capsule is 
stained a reddish color. This is known as McFadyeans color test, 
and if the specimen is properly, prepared is to be observed in all 
anthrax preparations made from dead animals. The test is most 
successf-ul in those preparations where the bacilli are very numerous, 
and could be of great importance in field work where a microscope 
may not be at hand. 

CULTURAL AND MORPHOLOGICAL CHARACTERS. 

The bacillus of anthrax has such definite and constant cultural and 
morphological characters that it can hardly be mistaken for any 
other bacterium. ^Ylien growing on the surface of agar-agar plate 
cultures beautiful colonies occur, appearing as wavy wreaths resem- 
bling locks of hair or bits of raw cotton thrown on the surface. It 
is supposed that the whole mass consists of thousands of contiguous 
cells forming one long chain, wound up in all directions. 

The growth in gelatin tubes is characteristic in that it liquefies the 
medium and the growth resembles an inverted fir tree. 

In milk a small amount of acid is formed, so that milk is slightly 
coagulated and slowly pe^Dtonized. 

In peptone bouillon virulent bacilli make a much more luxuriant 
growth than do the attenuated bacilli or vaccines. The heaviest 
growth is near the surface, where it can obtain oxygen, and consists 
of a dense mass of intertwined bacilli in chains which readily fall 
to the bottom of the tube if it be even slightly shaken. This heavy 
gTowth does not occur when bouillon is inoculated with attenuated 
bacilli. 

Growth on all media proceeds best at 35° C. The minimal tempera- 
ture for growth is 12° C., and the maximal 45° C. The vegetating 
bacillus is killed by a short exposure (15 minutes) to 60° C. moist 
heat, and by desiccation in a few days at ordinary temperatures. The 
spores, on the other hand, will stand almost any disinfecting process 



5 AITTHKAX. 

that any infected material can stand without greatly changing its 
physical characters. Hence it is impossible to disinfect hides con- 
taining anthrax spores without spoiling them for leather making. 
Spores require a boiling temperature in moist heat, and a much higher 
temperature in dry heat to destroy them. Spores live for a long 
time — probably 10 years — in the soil or in water, and it is because 
of this great resistance that pastures once infected remain so for an 
indefinite period. 

The bacillus can grow either in the presence or absence of oxygen, 
but it gTows best when air or oxygen is present. Spores can form 
only in the presence of oxygen, and this accounts for the fact that 
the bacillus never sporulates in the living blood nor in cultures gTown 
under anaerobic conditions. 

When spores begin to form, a tiny bright spot appears in the center 
of the parent cell, which soon increases to the diameter of the cell 
and always remains in its center. The cell protoplasm seems to be 
used up in the production of the spore, which now forms the resting 
stage of the organism. This spore remains quiescent until it is again 
placed in a suitable environment for growth, when it sprouts and 
assumes the bacillary or infective stage. 

Although the spore, or resting stage, of Bacillus anthracis is very 
resistant to all natural and artificial disinfectants, the bacillar or 
infective stage is quite vulnerable. The bacilli locked up in the air- 
less blood vessels or cavities of the body not only do not spore, but 
rapidly disintegrate after death. These important facts are of incal- 
culable value in the suppression of the disease by nature, and furnish 
the strongest kind of an argument for the prompt burial of animals 
dead from the disease. If such an animal be opened, however, the 
above-mentioned natural protection against the spread of the infec- 
tion is removed, as the bacilli will continue to multiply and sporula- 
tion will finally ensue. 

The following experiments were made to demonstrate the foregoing 
facts: The spleen of a guinea pig just dead from anthrax was minced 
and placed in a Petri dish exposed to air at room temperature. 
Microscopic examinations of this material 16 hours after showed that 
every bacillus had spored, the protoplasmic cellular material staining 
poorly. Another experiment made to determine the effect of such 
exclusion of the air as burial can afford showed that 24 hours are 
required for spore formation. 

In order to determine the effect of total exclusion of air upon the 
question of viability of the bacilli, capillary glass tubes were filled 
with heart's blood from a mouse just dead from anthrax and sealed 
in a flame. These sterile tubes were filled under precautions which 
prevented the ingress of extraneous bacteria; hence there were only 
anthrax bacteria in them, and the disintegrating effects of post- 



CHANNELS OF INFECTION. 9 

mortem bacilli through the septic ferments which they form in a 
decomposing carcass were not present. Examinations and cultures 
from these tubes were made at intervals up to the twenty-third day, 
and while the bacilli were lessened in numbers, they were still viable. 
In buried carcasses, even though they be not opened, putrefactive 
bacteria grow very rapidly and their excretions cause a granular 
degeneration of the anthrax bacilli. If air is admitted to the carcass 
these excretions have no deleterious effect upon the spores that have 
formed, and such a carcass infects the soil in which' it is buried, 
while if the carcass had not been opened, and care had been taken 
to disinfect the soiled exterior and air-containing cavities of the 
animal, the putrefactive bacteria would kill off the anthrax bacilli. 
The length of time necessary for this destruction varies with the 
temperature, being about a week in hot weather. 

CHAUITELS OF UTEECTION. 

There are four routes by which animals may become infected, 
namely, by ingestion, by inoculation, by inhalation, and by trans- 
mission. 

Ingestion. — The first and most frequent, and therefore the most 
important, is by ingestion. This form is acquired when animals are 
turned out to pasture. 

Inoculation, — The method of infection next in importance is by 
inoculation, which is brought about by contact of abrasions and 
wounds with infected material, or by flies or other insects. 

A case of spontaneous inoculation arising in the course of my ex- 
periments — the only one which occurred in the work — was where a 
ram, immune to anthrax, was Severely bled to furnish blood for 
experiments. He was kept in a stall where a sheep had died three 
months previously of experimental anthrax. A considerable wound 
was made by the operation and the animal was found dead of an- 
thrax on the second day after having been bled. 

Inhalation. — The form third in importance is by inhalation. Here 
spores are breathed in with dust arising from infected material or 
surfaces. 

Transmission. — A fourth form, which is probably rare, is that ot 
transmission from the mother to the fetus in utero. I have observed 
this in the case of an experimental cow which had been immunized 
with a single anthrax vaccine and subsequently tested with virulent 
bacilli. The m_other at no time appeared sick, but on the sixteenth 
day after receiving the virulent bacilli she dropped a dead calf, 
which appeared to be a full-term one. Not suspecting the cause of 
the trouble, the calf and membranes were buried. Fortunately, a 
loop of the discharge found on the ground was plated and developed 
a pure cultivation of 60,000 anthrax bacilli, the virulence of which 
89049'^— BuU. 137—11^^ 3 



10 ANTHRAX. 

was demonstrated on a guinea pig. The cow was in no way sick, 
and daily microscopic examinations of the vaginal discharges showed 
these were free of anthrax bacilli. 

INFECTION OF THE SOIL. 

Soil becomes infected with anthrax by discharges from animals 
suffering with the disease, and by the dead bodies of animals which 
are allowed to decompose upon the surface. These discharges con- 
sist of small hemorrhages which occur from the mucous membranes 
in the nose and bowels, mixed with the nasal secretions and with 
the feces. These sometimes do not occur until just before death. 
In some cases the discharges are so limited as almost to escape obser- 
vation, but a microscopic examination in such cases would reveal 
large numbers of bacilli. Hence it is very important to limit to the 
smallest area possible the wanderings of an animal so affected. It is 
doubtful if the feces of the smaller experimental animals contain 
anthrax bacilli. One observer reports the presence of anthrax spores 
in the feces of guinea pigs, but I have never found them. 

Pasteur, in his memorable paper on anthrax before the Academy 
of Sciences in Paris in 1880, gave his opinion as to another manner in 
which the surface becomes infected with this disease. He said 
in part : 

Earthworms are the bearers of the germs, and they bring this terrible para- 
site to the surface of the ground from the depths to which it has been buried. 
It is in the little cylinders of earth, of fine earthy particles, that the worms ex- 
crete and deposit at the surface of the ground after morning dews, or after 
rains, that are to be found, besides a crowd of other germs, the germs of 
charbon (anthrax). 

While it does not seem impossible that earthworms may swallow 
the spores of anthrax and cast them out upon the surface, that close 
observer, Koch, was unable to verify the facts. He held that pas- 
tures remain infected on the surface from preexisting cases, and 
that the parasite remains on or near the surface from season to 
season and infects animals which graze upon such soil. When the 
grass gets short in dry weather, spores that may have been carried 
into the soil by previous rains either rise again by capillarity or are 
brought to the surface by the animals pulling the grass up by the 
roots. The writer has examined many earthworm casts from an- 
thrax graves of various ages — from 15 years to 1 month — and has 
failed to find anthrax spores in them. Pasteur's work was done be- 
fore the days of solid media, and hence the plate-culture method of 
isolation was not used by him. Another disease, malignant edema, 
is easily produced in guinea pigs and rabbits, and greatly resembles 
anthrax clinically, as well as in the morphology of the organism 



METHODS OF DIAGNOSIS. 11 

causing it. These two germs would greatly resemble each other 
when viewed with a microscope made in 1880. 

In order to test further the question as to whether the surface above 
an anthrax grave becomes infected by earthworms, or otherwise, the 
writer has for the past three years pastured sheep and cattle upon 
soil in which there are many anthrax graves, and no case of spontane- 
ous anthrax has arisen. The animals were frequently observed graz- 
ing in the little hollows formed by the sinking in of the graves, and 
it would seem that this is sufficient proof of the questionableness of 
Pasteur's early theory regarding this source of infection. 

METHODS OF DIAGNOSIS. 

Anthrax is a disease that we absolutely know is caused by the para- 
site ascribed to it. We can tell just how long an animal will live after 
it has been inoculated with a given culture, provided the culture is of 
maximum strength. We know that a virulent culture will, in proper 
dose kill the laboratory animals, as well as sheep, in 48 hours, and 
that we can always recover the germ in the blood of such animals 
and reproduce the same disease in another animal b}^ inoculating it 
with this blood. Hence, all of Koch's postulates can be fulfilled. 

Different races of anthrax bacilli vary in virulence. Those of 
highest virulence will kill guinea pigs and rabbits in 0.00001 c. c. 
doses of a 24-hour bouillon culture in 48 hours, and the same culture 
would kill a sheep in 95 hours when given a 0.0001 c. c. dose subcu- 
taneously. Very young animals are much more susceptible to anthrax 
than adults, and hence care must be exercised in vaccinating young 
animals. A sucking rabbit a few days old died in 18 hours from a 
0.1 c. c. dose of bouillon culture, and the virulence of the bacillus, 
which had become somewhat lessened, was greatly enhanced. 

A diagnosis can be made by the veterinary scientist by two meth- 
ods ; either by a bacterioscopic examination of blood smears made soon 
after death or by animal inoculations. On account of the nature of 
the disease it is highly desirable that the first method be used, as no 
time should be lost in coming to a decision. It is easy to reach a de- 
cision if the specimen has been properly prepared and if the micro- 
scopist is familiar with the organism. 

PREPARATION OF SPECIMENS. 

A small drop of blood thinly spread on a piece of glass or paper 
and dried immediately is all that is required. It is best to obtain 
blood for this purpose by making a needle puncture into the jugular 
vein, as sometimes the bacilli are not numerous in the peripheral 
blood vessels. A specimen prepared in this way will arrive at the 
laboratory just in the condition in which it was collected. There 
will be no bacteria in it that were not there when it was prepared. 



12 ANTHRAX. 

Sometimes specimens arrive consisting of two smeared surfaces of 
glass stuck together. These are dangerous, and it is next to impos- 
sible to separate them. They should be boiled and thrown away, no 
attempt being made to make an examination. 

The most dangerous anthrax package, and one which is not alloAved 
in the mails, is the bottle of blood. These have arrived broken, and 
in one instance a positive diagnosis was made from blood smeared on 
the outside of the package by the sender's hands. Again, when parts 
of organs, or blood in volume, are sent in, decomposition will have 
taken place and a diagnosis may be impossible. The fruit-jar pack- 
age,, containing spleen, liver, heart, etc., sent by express, is another 
most dangerous package. These have arrived broken, with liquid 
dripping from them. When not broken their opening is generally 
attended with dangerous difficulties. The top is usually screwed 
down so tight that considerable force must be used to open it, and 
when this is accomplished the gases which have- formed will spray 
the liquids upon one's hands and clothing. 

A diagnosis of a blood-smear preparation is made b^^ first " set- 
ting " the specimen by heat, passing the glass slide through the flame 
three times, occupying a second each time. The specimen is now 
stained with methylene blue or fuchsin, dried between two pieces of 
filter paper, and mounted for examination. If anthrax bacilli are 
present they can be easily seen stained blue or red, according to the 
stain used. They appear as rods with square ends. They are never 
round or blunt. They will almost alwaj^s appear in chains, although 
they frequently appear as single cells when torn apart in making the 
preparation. Spores are never found in a specimen prepared from a 
recently dead animal. With lenses of 1,000-1,200 amplification the 
ends appear somewhat concave, from bulging of the corners of the 
cylinder. When such an organism is found in the blood of an animal 
recently dead the disease may be called anthrax, as it is the only one 
in animals that provides such a picture. 

ANIMAL INOCULATIONS. 

When there is reason for doubt, animal inoculation must be re- 
sorted to. Mice, guinea pigs, and rabbits may be used, and these are 
susceptible in the order named. The suspected material is ground 
up in a sterile mortar containing normal salt solution. The solid 
particles are allowed to settle or are filtered off, and a few drops to 
0.5 c. c. of the liquid are injected subcutaneously into guinea pigs. 
If anthrax be present the animal will die in two or three days, and 
the bacillus can be recovered from its blood and examined as in the 
first method. Plate cultures should also be made from the extract 



DIFFERENT FORMS OF ANTHRAX. 13 

of the suspected substance, as it is possible to isolate the anthrax 
organism and thus come to a decision from a study of the anthrax 
colonies if any develop, as such colonies have a characteristic appear- 
ance. Inoculation into experimental animals of cultures made from 
these colonies, if they be anthrax, will cause the disease, and thus any 
doubt as to their identity will be removed. 

DIFFERENT FORMS OF ANTHRAX. 

Anthrax manifests itself in different forms according to the seat of 
invasion, kind of animal, virulence of the bacillus, season of the year, 
and time of the outbreak. 

The most acute type of the disease is seen oftenest in cattle and 
sheep and is known as apoplectic or fulminant anthrax. This form 
appears suddenl5^ without premonitory symptoms. The animal is 
suddenly seized with trembling, a haggard expression, swaying, diffi- 
cult breathing, and cyanosis. Convulsions and death occur in from 
a few minutes to two or three hours. Bloody discharges usually 
occur during the last hour from the nose and rectum. These are 
very infectious, as the bacilli are present in large numbers, and 
constitute an important way in which pastures become infected. 
For this reason such animals should be confined where these dis- 
charges can be disinfected. 

The second form, known as anthrax fever or internal anthrax, 
differs from the fulminant or apoplectic form only in its duration. 
The symptoms in the last stages are practically those seen in the 
most acute type, except that they are more intense and of longer 
duration. There will be excitability, restlessness, high fever, oozing 
of blood from the nose, eyes, ears, rectum, and thinner parts of 
the skin of the axilla or thigh ; tremors, dullness, prostration, grind- 
ing of the teeth, colicky pains, difficult breathing, arching of the 
back, rolling of the eyes, convulsions, and death in sheep in 24 
hours, in ^attle in from 2 to 5 days, and in horses in from 1 to 5 
days. 

The third form is local or external anthrax. In this form the 
disease is at first localized in the mouth, throat, or skin in cattle, 
and the same form appears most often in horses in the tongue, 
throat, neck, breast, withers, shoulders, flank, or thigh. These 
swellings are firm or doughy, insensitive at certain parts, and tend 
to become gangrenous. There is no tendency to suppuration, and 
they do not crackle on pressure — a diagnostic point when there is 
doubt as to whether the case is one of anthrax or of blackleg. When 
cut into — something which should never be done, as the exudation 
always contains the anthrax bacillus— there escapes either a pale, 
straw-colored, or bloody liquid. 



14 AKTHEAX. 

SYMPTOMS. 

The symptoms of anthrax will vary with the species and with the 
type of the disease, except that in the last stages of any of the three 
types the most pronounced symptoms are identical. In the most 
acute type the animal may appear at first to be perfectly well and 
keep along with its fellows even when its temperature is very high — 
as high as 106° F. Along with such a temperature we shall of course 
find a rapid pulse and increased respiration. Wlien one is standing 
close beside such an animal the heart beats may be plainly heard. 
Soon other symptoms, such as grinding of the teeth, tremors, and 
standing with head down, appear. Then appear drooping of the 
head and ears and-, a disposition to lie down. Animals that have been 
lively will now decline to rise unless 'handled roughly. They become 
stupid and sleepy and very weak in the hind parts. Whereas the 
temperature has all along been high, it now shows a sharp decline, 
and before death may become subnormal. The visible mucous mem- 
branes are a dusky red, especially those of the rectum and vulva. 
There is a bloody nasal discharge. The feces will be coated with a 
bloody mucus. Local swellings appear in the mouth, throat, neck, 
and breast (especially in horses), and there are sharp attacks of colic 
and convulsions which end the misery of the animal in from 12 to 48 
hours after the disease is first noticed. Pregnant animals are liable 
to abort and thus greatly spread the infection through the copious 
discharges. An outbreak has its highest mortality at its onset, while 
later on some animals, especially horses and mules, may recover. In 
the most acute types, which occur mostly in cattle and sheep, the 
animal is found dead. A cow which seemed well at night is found 
dead in the morning, or if death occurs in the daytime, the illness is 
of short duration, occupying only a few minutes or' one or two hours. 
In these sudden attacks the symptoms follow each other so rapidly 
and death is so sudden that sometimes the owner is convinced that 
the animal has been poisoned. The attack is ushered in with trem- 
bling, anxious expression, high fever, rolling of the eyes, and con- 
vulsive movements, soon followed by general convulsions and death. 

In the local form or cutaneous anthrax in cattle, swellings appear 
suddenly on different parts of the body at one or many places, and 
the animal dies with the same symptoms as occur in the most acute 
type when the bacilli reach the circulating blood from these local 
lesions. When the infection occurs in the tongue or pharnyx, we have 
in the first case gloss anthrax, and in the latter pharyngeal anthrax, 
the symptoms varying somewhat according to the part most affected ; 
but the general constitutional symptoms will be those already de- 
scribed. In some cases the most prominent symptoms at first will 
be enormous swellings of the rectal mucous membrane. 



POST-MORTEM APPEAEANCES. 15 

In the local form, or cutaneous anthrax, in the horse, the swellings 
occur at the points of entrance of the bacilli or spores, where there 
are abrasions of the skin or mucous membrane, or where biting in- 
sects have brought the infection from a previous case. These swell- 
ings appear suddenly at the point of inoculation and are character- 
ized by a rapidly spreading edema. The general symptoms are not 
so urgent, the fever is less intense, and the mortality, while not so 
great as in the more acute form, is still high. 

POST-MORTEM APPEARANCES. 

An animal that has died of anthrax will nearly always be found 
much bloated, with blood oozing from the nose and rectum. There 
will be evidences on the ground that the animal died a violent death, 
in convulsions. Local swellings will be present or absent according 
to the type of the disease. In the rapidly fatal cases little change 
will be noted either in the blood or internal organs beyond those 
produced by high blood pressure, indicated by a swollen spleen and 
engorged liver. The carcass itself — the edible portions — would 
show nothing that would make it doubtful as food. In the more 
prolonged cases a hemorrhagic condition will be noted in all the 
internal organs. The blood will be tarry in appearance and will 
not clot. The heart is often light in color, while on the inside it 
will be found deeply stained and containing dark, uncoagulated 
blood. The liver may be found enlarged and is easily torn in han- 
dling, presenting on its surface hemorrhagic areas. The spleen is 
often specially enlarged and distorted in shape, and ruptures on 
handling. 

The bacilli of anthrax can be found in largest numbers in those 
organs where the lesions are most pronounced, namely, the spleen, 
liver, and engorged lymphatic glands, but they are commonly found 
in any part of the vascular system. In the serous cavities, such as 
the pericardial, the pleural, and the peritoneal, may be found a san- 
guineous fluid consisting of serum, red and white cells, and anthrax 
bacilli. The hyperemia and areas of extra vasated blood may appear 
at any point of the body where the bacilli have become localized and 
enormously multiplied, forming capillary embolisms, which consist 
of broken-down blood corpuscles, and bacilli. Hence they are fre- 
quent in the tongue, throat, lungs, stomach, and intestinal walls ; the 
mesentery and the omentum; the skin, connective tissue, and the 
muscles. 

THE CAUSE OF DEATH. 

From the manner of death it would seem that the poison of an- 
thrax acts specifically upon the center of respiration, this in turn 
allowing a fatal accumulation of carbon dioxid in the blood. That 



16 ANTHKAX. 

it is the replacement of the oxygen of the blood by carbon dioxid that 
is the immediate cause of death in anthrax is further shown by the 
loss in the blood of the property of coagulation, it being known that 
carbon dioxid precipitates and throws out of action the fibrin-form- 
ing element of the blood — fibrinogen. 

DISPOSAL OF CARCASSES. 

The disposal of an anthrax carcass should not be left to the owner 
nor should anyone be allowed to open a carcass of an animal that is 
supposed to have died from anthrax. The diagnosis can only with 
certainty be made by a competent bacteriologist. He need only be 
furnished a drop of blood from the suspected animal. This blood 
can be drawn from the jugular by means of a syringe, or a small nick 
sufficient to allow a drop to escape may be made with a suitable in- 
strument. This blood should be smeared upon a piece of glass or 
piece of paper, dried immediately, and forwarded to the bacteriolo- 
gist. Any blood escaping during the above operation should at once 
be disinfected. The State should appoint a sanitary agent in every 
election district, whose duty it should be to dispose of anthrax car- 
casses, according to instructions issued by a competent authority, as 
it is only by the proper disposal of the carcass and by vaccination that 
the disease can be controlled. While the majority of our farmers 
recognize the advisability of promptly burying the carcass, there are 
those who would allow the animals to lie where they die to decompose 
or be eaten by dogs and buzzards. Parts of the carcass may be car- 
ried by dogs to adjoining or distant pastures and spread the infection. 
Spores forming in the carcass opened in this way would pass through 
the intestinal canal of buzzards and dogs and permanently infect 
pastures miles away. The soil upon which such an animal has lain 
would certainly be infected and remain so for many years. 

During the last few years deep burial, rather than efforts at crema- 
tion, has been practiced, with good results so far as can be ascertained. 
While cremation properly carried out is undoubtedly the safest 
method of disposing of an anthrax carcass, it is practically impos- 
sible to destroy absolutely all vestiges of the body and bacilli-laden 
body fluids, and unless this is done the whole operation so far as it 
goes is almost a useless waste of time, material, and labor. In the 
cremation of carcasses in the field even the earth upon which it has 
lain should be thoroughly and deeply burned over so that the heat 
will penetrate to a depth sufficient to insure the death of the anthrax 
bacilli which certainly passed into the soil with the body fluids when 
rupture occurred from the heat. We know enough of the biology of 
the anthrax bacillus to take advantage of the fact that if it is kept 
locked up in the closed cavities and blood vessels of the body where 
no air can reach it, not only will the bacilli fail to produce the re- 



DISPOSAL OF CAECASSES. 17 

sistant spore, but they will actually decompose and become noninfec- 
tious, so that the bacilli which are in the blood vessels of an unopened 
animal perish. On the other hand, those bacilli which have come 
away in the pulmonary and rectal discharges during life will, if they 
are provided with sufficient material to keep them moist, promptly 
go into the spore or resting stage. These constitute the main source 
of danger, and in this way a considerable area may become infected. 
For similar reasons, hemorrhages into adr-containing cavities, such as 
the lungs, trachea, head, sheath, vagina, and rectum, all favor the 
production of the resistant spore. Hence, if we introduce into the 
cavities a disinfectant which will surely kill the spores, and also at 
the same time take care to disinfect the soiled exterior surfaces of 
the animal, and then bury such an animal without opening it, we may 
reasonably conclude that such a carcass will not thereafter be a 
source of infection. 

THE OFFICIAL METHOD IN DELAWARE. 

In Delaware the method of procedure as advised and as carried out 
by the State board of agriculture is as follows: Formalin is injected 
in both directions into the trachea. Formalin-soaked cotton is pressed 
up the nostrils and into the mouth, rectum, vagina, or sheath. The 
grave is dug where the animal dies, when possible. The body of the 
animal is then either thoroiighly wetted down with a 4 per cent solu- 
tion of formalin or kerosene, or coal oil is poured over the uppermost 
side of the animal and is set on fire. Only enough kerosene is applied 
to burn off the hair, as it would defeat the purpose to burn the animal 
sufficiently to cause rupture of the body. The carcass is then rolled 
into the grave with the other side uppermost. This side is now 
sprinkled with the oil and fired, after which the dirt upon which the 
animal has lain is disinfected w^ith formalin and is then shoveled into 
the grave first. The grave is now filled and banked up as is the cus- 
tom. Such graves should be surrounded by a fence. In fact, there 
should be a graveyard widely fenced in, and when an animal is known 
to have anthrax, it should be placed therein and allowed to die where 
it is to be buried. Such an inclosure should never be used for any 
other purpose, and should be so located that drainage from it upon 
other land will not occur. 

SUSCEPTIBILITY OF THE VARIOUS ANIMALS. 

While all the farm animals and even man himself will contract 
anthrax, we know that the degree of susceptibility varies not only in 
the different species, but also in individuals of the same species. We 
find that the herbivora are very susceptible and that the carnivore 

89049°— Bull. 137—11 3 



18 ANTHEAX. 



■sx. 



are less so, while the omnivora occupy a place between these two 
extremes. Cattle and sheep, being extremely susceptible, contract 
the fulminating type in nearly every case, while horses and mules 
frequently live several days, exhibiting the local or skin form, and 
some cases recover. It could probably be shown that any ruminant 
is more susceptible than other herbivora because of the anatomical 
and physiological characteristics of their respective digestive organs. 
In ruminants we find the immense rumen, with its moisture, heat, 
and alkaline reaction, an admirable incubator for bacteria. The out- 
ward passage of the contents of the rumen is slow, so there is plenty 
of time for anthrax spores swallowed with the food to develop into 
bacilli and multiply into prodigious numbers before they pass through 
the other two stomachs into the true or acid stomach, where some of 
them may be digested, but where many may pass out unharmed into 
the alkaline intestine and be absorbed with the food, finally entering 
the blood stream suspended in the chyle. It would now require only 
a few hours for them to multiply in the blood into sufficient numbers 
to produce their fatal poison. It is doubtless in this way that cattle 
which seem well when last seen and fed at night come down suddenly 
with the disease and are found dead in the morning. 

Swine are less susceptible than horses and cattle, yet when swine 
eat the carcasses of animals dead from anthrax they may contract 
the disease. As in other species, the young sucking pigs are more 
susceptible than the adults. As swine usually contract the disease 
by eating the dead animals, they generally have the disease in the 
throat or intestine. The symptoms in such cases do not vary 
materially from like cases in the other species. 

Dogs and cats contract the disease similiarly to hogs and are 
classed among the less susceptible. The symptoms are those to be 
expected, and consist of swelling of the throat, difficult swallowing, 
vomiting, profuse bloody diarrhea, high fever, and death. 

Birds of prey are said to be immune to the disease. Chickens are 
less susceptible than swine, yet Pasteur showed that chickens were 
quite susceptible when immersed in cold water and then inoculated. 
Likewise frogs, which are ordinarily immune, can be successfully 
inoculated after immersion in warm water. Young pigeons of 
certain breeds are easily infected artificially, and sparrows, finches, 
canaries, yellow-hammers, and redbreasts have been successfully 
inoculated. 

In chickens anthrax runs a very rapid and fatal course in 24 
hours. They can contract the disease by eating the carcass or dis- 
charge of an animal dead from anthrax. As in other animals, a 
differential diagnosis between this disease and other chicken diseases 
of like type should be based upon the discovery of the anthrax 
bacillus in the blood along with the symptoms of sudden debility 



ANTHRAX IN MAN. 19 

and high fever. Anthrax swellings will occur on the comb and 
wattles, around the eyes, and on the tongue, palate, and feet. The 
bird is extremely weak, has violent tremors and convulsions, and 
bloody diarrhea, and dies within 24 hours. 

ANTHRAX IN MAN. 

Anthrax is not, strictly speaking, a human disease, yet it is not 
rare in the practice of physicians who attend the workers in tanneries 
where imported hides are converted into leather, nor is it rare in per- 
sons whose occupation brings them in otherwise contact with hides, 
wool, hair, furs, hoofs, bones, rags, felt, glue, or any product made 
from the bodies of animals that die of anthrax. Hence the disease is 
found in shepherds, cattlemen, horsemen, farmers, drovers, butchers, 
tanners, brush makers, and veterinarians. 

The disease is notoriously common in those who handle imported 
hides, although man can become infected through the bites of flies or 
other infected insects. The sound skin is sufficient protection against 
the entrance of the bacillus, but the slightest abrasion is sufficient as a 
point of inoculation in handling infected material, or even for inocu- 
lation by the ordinary housefly that has visited the discharges of an 
animal suffering with anthrax. The process of tanning does not 
alwavs disinfect an anthrax hide. That hair is a medium of infec- 
tion is shown by outbreaks of anthrax among brush makers. Per- 
sons who handle imported wool and rags contract the disease by in- 
haling the spores in the dust which arises. This spore develops into 
the bacillus, and a case of pulmonary anthrax is established. This 
form is known as w^oolsorters' disease, or ragpickers' disease. Those 
who handle bones and carcasses in fertilizer, glue, and rendering 
establishments located in anthrax districts are peculiarly exposed to 
infection. In man the large majority of cases occur on the face, prob- 
ably because this part of the body is more liable to be attacked by 
biting insects. 

Anthrax in man is a local disease, and we do not find the bacilli in 
as large numbers in the blood or other organs, such as the liver, 
spleen, and kidneys, as we do in the lower animals. The bacilli can, 
however, be found in abundance in the local lesion. 

MALIGNANT PUSTULE. 

Malignant pustule, or local anthrax of the skin, usually occurs on 
the face, hands, arms, or neck of those who handle imported hides. 
In from one to three days after infection takes place a small red 
pimple appears. This soon changes to a vesicle, which is very pain- 
ful. The center of the vesicle rapidly becomes necrotic, forming a 
black eschar, which soon becomes surrounded by a ring of vesicles. 



20 ANTHRAX. 

The surrounding tissues become congested and edematous, and the 
lymphatics are involved. The fever is quite high, and the patient 
is very sick from the absorption of poison from the local disease. It 
runs a rapid course, and the patient may die from toxemia. 

Most of these cases are, fortunately, susceptible of successful treat- 
ment. This consists of early and thorough excision of the pustule 
and all infected surrounding tissue, followed by the local application 
of strong disinfectants. At Guy's Hospital, in London, 13 out of 15 
cases were cured by excision, even though in 12 of these cases the 
inflammation had spread to the surrounding parts or had involved 
the lymphatic glands with more or less severe constitutional disturb- 
ance. In some cases the disease process goes no further than the 
formation of the eschar, which then becomes- a scab, with subsidence 
of the inflammatory process, and recovery. In the majority of cases, 
however, the disease runs a regular course; if not excised early the 
bacilli enter the blood stream and the case terminates fatally from a 
modified form of the same disease as occurs in cattle, with the excep- 
tion that the bacilli are not found in large numbers in the internal 
organs nor is the spleen greatly enlarged. It may, therefore, be said 
that in man death is largely due to the absorption of toxins from the 
local lesions. When the pustule is situated on the extremities the per- 
centage of recoveries under treatment is large. 

PULMONARY AND INTESTINAL ANTHRAX. 

Pulmonary anthrax, or woolsorters' disease, is caused by the inha- 
lation of spores by those who handle hides or other infected material. 
The initial lesion is local and is situated in the lower trachea. It 
consists of a swollen and hemorrhagic condition of the mucous mem- 
brane, with great enlargement of the mediastinal and bronchial 
glands and of effusions into the pleural and pericardial cavities and 
the lungs. Externally may occur also cutaneous edema over the 
chest and neck and inflamed glands. This form is rapidly fatal, the 
patient dying from suffocation and toxemia. 

Intestinal anthrax is also of local origin and has about the same 
pathology and terminations as the pulmonary form. There is intense 
inflammation of the intestinal mucous membrane and involvement of 
the neighboring lymphatic glands. Intestinal infection takes place 
when anthrax-infected products are eaten. 

THE ANTHRAX SEASON. 

Anthrax is peculiarly a disease which may be said to be a seasonal 
one; that is, the disease makes its appearance with the advent of 
certain kinds of weather. In March, 1907, in Delaware we were 
visited with some exceptionally warm, springlike weather of con- 



THE ANTHRAX SEASON. 21 

siderable duration, and on the 22d of that month the first case oc- 
curred for that year, an exceptionally early start. In 1908 the first 
case occurred on May 2. In 1909 the first case did not occur until 
July 23, but the disease persisted until November 6, whereas in 1907 
the last case was on September 24, and in 1908 on August 24. So 
that we may say the season lasts, in Delaware, from March to No- 
vember, being most prevalent in the summer months. As the disease 
is, generally speaking, a warm-weather one, and as we know that 
the causative agent is present throughout the year, there must be 
some conditions which come with warm weather which predispose 
to the onset of the disease, either by affecting the receptivity of the 
animals or by increasing the infective properties of the germ of the 
disease. Possibly both conditions prevail. There can be little doubt 
that the resting stage of the Bacillus anthracis — the spore — ^will 
quickly develop into the bacillar or infecting stage under the influ- 
ence of heat, moisture, and organic matter. These conditions pre- 
vail on the pasture in warm weather, and the writer has demon- 
strated that the germ of anthrax will vegetate in a 2 per cent hay 
infusion. Hence, when spores of anthrax come in contact with hay 
infusions in pastures, especially in meadows, there is produced a viru- 
lent culture which will infect the animal eating them. 

The animals themselves are probably more susceptible to anthrax 
when turned out upon fresh pastures, as the ingestion of rank grasses, 
which are acid in reaction, lessens the normal alkalinity of the blood 
and thereby increases the susceptibility to anthrax. This greater 
alkalinity of the blood in carnivora may be the cause of their greater 
resistance to the disease, while the loss of alkalinity of the blood by 
the herbivora, and especially by ruminants such as cattle and sheep, 
through the ingestion of such large quantities of acid grass, may 
account for their increased susceptibility during the grass-growing 
season. At all events there are certain weather factors which de- 
termine, in a measure, the onset of the anthrax season, and given such 
conditions the season can be predicted with tolerable certainty. This 
season exists when we have had a long, dry period, followed by light 
rains or infrequent heavy rains, and then by extreme heat. This kind 
of weather will not only produce bacterial' multiplication, but will 
cause a rapid growth of rank acid grasses, and these when eaten 
will increase the susceptibility of animals to anthrax by decreasing 
the alkalinity of their blood. 

Anthrax, then, may be said to be a pasture disease, wliich exists 
mostly in warm weather. If it were a stable disease, why should we 
not have cases occurring in winter, and why is it that the disease 
rarely occurs in animals that are not turned out on pasture? We 
rarely see a case of anthrax in a carriage horse that is fed in the 
stall, but it is not of infrequent occurrence in horses that are turned 



22 ANTHRAX. 

out to pasture. Cases are rare in city horses, but common in the 
farm horse. The city horse must eat the same hay and grain, gener- 
ally speaking, as the farm horse. If it were the hay and grain that 
produce the disease, we should find anthrax as common in city stalls 
as on the farm. 

Anthrax rarely occurs in small towns. It is distinctly a disease 
of animals that graze on infected fields. A recent case emphasizes 
this. An expressman delivered some goods to a farmer who lives 1 
mile from this town (Newark, Del.). This farm has anthrax every 
season. The expressman's horse must have grazed upon infected 
soil while there, as it was sick within 48 hours with high fever and 
edematous swellings at the root of the neck and down the front legs. 
A microscopic examination of the exudate showed the presence of 
anfehrax bacilli, and the. animal died in a few hours. This animal 
lived in the stable with others, and was fed the same feed. No case 
has occurred since, and it was the only animal from that stable that 
visited the infected farm. The great importance of an early diag- 
nosis and isolation was demonstrated in this case. Within a few 
hours after a swelling was noticed anthrax was suspected and some 
of the bloody subcutaneous exudate was aspirated and brought to the 
writer by the attending veterinarian for diagnosis. It was thus 
possible to remove the animal from the stable to a yard where other 
animals could not become infected long before any infecting dis- 
charge had taken place. The animal was embalmed, washed down 
with formalin solution, as previously described in this article, and 
buried in a six-foot grave as soon as the same could be dug. The 
stables have been used uninterruptedly ever since (four months), 
and no fresh case of anthrax has occurred. 

PREVALENCE OF ANTHRAX IN DELAWARE. 

Anthrax has been officially recognized as existing in this State 
(Delaware) since August, 1892. It is highly probable that the disease 
had its origin in imported hides used in the tanneries at Wilmington 
for making leather. Large quantities of scraps accumulate during the 
process of leather making, and these were sold to farmers who com- 
posted them and later spread the infected material upon the land. 
Since the spores of anthrax would live and retain their virulence in 
a compost heap, it is easy to see how the lands first became infected. 
While it is generally conceded that meadow pastures are most pro- 
ductive of the disease, the tillable lands were probably infected first, 
as above stated. The meadow pastures haA^e retained their infective 
properties because of their constant use for grazing, and particularly 
because of fresh cases of anthrax occurring upon them. The higher 
tillable lands have, however, in many cases become disdnfected by the 
lapse of time, cultivation, ajid drainage. 



PREVALENCE OF ANTHRAX IN DELAWARE. 23 

Estimates made by veterinarians practicing in the State show that 
there are from 175 to 200 farms in Delaware that are permanently 
infected with anthrax. These farms are located, in a general way, 
in that territory which drains into the Delaware River and the Dela- 
ware Bay. The infected territory comprises about one-third of the 
total area of the State. 

While anthrax has been known to exist in the State only since 
1892, it is highly probable that the first cases occurred much earlier 
and that the disease is as old as the morocco-leather industry. The 
originally infected territory must have been the cultivated lands 
upon which infected compost fertilizer was spread by the farmer him- 
self. Animals dying on such farms before the nature of the disease 
was known here were either hauled out dead or turned out while yet 
alive upon the marshes to die. Doubtless they would in some cases 
be set adrift in the creeks and thus- infect any shore or marsh upon 
which they drifted. As the marshes are always wet and contain 
decaying vegetation in abundance, the conditions for the development 
of the microbe of anthrax are perfect, while upon the dry cultivated 
lands, even though the microbe be present, the conditions are, owing 
to the comparative absence of water, much more unfavorable to the 
multiplication of the causative agent. It is because of these condi- 
tions that marsh pastures are regarded as being more productive of 
the disease. The wash water from the tanneries at Wilmington can 
no doubt infect the streams into which it flows and cause the disease 
in animals either grazing upon them or drinking from them. 

Those farms and marshes now infected will remain so for many 
years, even though no new cases occur upon them. This liability to 
outbreaks of anthrax on a farm ha& a depressing effect, not only upon 
the value of the land, but upon all agricultural operations. 

While there has been no epidemic of anthrax in Delaware for 
several years, and the number of deaths from the disease has been 
exceeded by many ordinary diseases of which little notice is taken, 
anthrax must always be considered a menace to the agricultural wel- 
fare of the State because of the ever-present liability of an outbreak 
which might assume the proportion of a genuine epidemic. This 
liability should be offset by the annual vaccination of every farm 
animal in the infected territory. Even though several years may 
have elapsed since the last case occurred on a farm, that farm is 
liable at any time to be again visited by the disease. 



24 ANTHRAX. 

METHODS OF PRODUCING IMMUNITY IN ANIMALS. 
THE EXISTING METHOD OF DOUBLE VACCINATION (PASTEUE METHOD ). 

Since 1892 anthrax has been controlled by vaccination by a method 
devised by Louis Pasteur. This method consists in the subcutaneous 
injection of attenuated cultures of the bacillus of anthrax. Two in- 
jections of varying degrees of strength are made at an interval of 12 
to 14 days. The first injection consists of 1 cubic centimeter of a 
culture that has been incubated at 42° to 43° C. for a sufficient time 
to decrease its virulence to a point where it will kill white mice, but 
not guinea pigs or rabbits. This generally requires a period of 24 
days, assuming that the culture was made originally from a moist, 
virulent race of anthrax bacilli. Such cultures are to be made 
directly from the heart's blood of an animal that has died of anthrax 
within 48 hours after inoculation. In such blood we find only non- 
spore-bearing bacilli, and when these are grown at 42° to 43° C. they 
do not at any time produce spores while this temperature is main- 
tained. Contrary to the statements of some, however, the bacilli 
promptly form spores when this temperature is reduced. These 
spores do not acquire any more virulence, however, than the parent 
bacilli which produced them. 

The second injection consists of a culture similarly made and 
incubated at 42° to 43° C. for a period of 12 to 18 days, or one 
whose virulence has been reduced to a point where it will not kill 
rabbits but will kill white mice and guinea pigs, the latter in 3 or 4 
days. The author's experience has shown that there is no hard and 
fast line in the number of days that a culture of a given race of 
anthrax bacilli must be attenuated. Much depends upon the re- 
sistance of the bacillus, the character of the culture medium, the 
exactness of the temperature of the incubator, and the natural re- 
sistance of the animals used in testing the vaccines, ^^^en a culture 
of proper strength has been obtained and is property transferred 
to fresh media about once a month and kept in a cool place where 
it will not evaporate, it may be used indefinitely as a stock culture 
for inoculating a liquid medium, which constitutes the vaccine. AVhen 
such attenuated cultures are inoculated into animals, a very mild 
and clinically unnoticeable attack of anthrax is produced, which 
confers an active immunity which persists throughout the anthrax 
season. The inoculation must be repeated the following season. 

Certain precautions are necessary in the application of anthrax 
vaccine. Assuming that the vaccine has been properly prepared, -it 
is the duty of the veterinarian to ascertain that anthrax is not 
already existing in the animals he is about to vaccinate. As a pre- 
caution, when there is reason to suspect that anthrax may already 



METHODS OF PKODUCING IMMUNITY. 25 

be existing, no animal showing a fever should be vaccinated, as the 
disease may be carried from the already infected animal to others 
upon the point of the inoculating needle. Again, in order to prevent 
abscess in horses and mules b}^ introducing under the skin strepto- 
cocci or staphylococci by the point of the needle, the place of injec- 
tion should be disinfected of. these microbes. The anthrax bacillus 
can not produce pus, but the germs usually found upon the skin 
are to be held responsible when abscess occurs from vaccination. 
The writer has been in the habit of dipping the point of the needle 
into strong carbolic acid contained in a small vial which may be 
conveniently carried in the side pocket at the time of vaccinating. 
None of this acid can enter the needle and kill the vaccine germs, 
as it is already filled with the vaccine, and none can be introduced 
under the skin, as it is all removed from the needle in its passage 
through the skin, and this in turn disinfects the wound made. While 
every veterinarian knows that hypodermic injections of medicine 
are daily made with a minimum of abscess production, it must be 
recognized that the conditions are not similar, and hence it is deemed 
highly advisable that these precautions be taken, especially in horses 
and mules. 

There is usually little or no swelling at the point operated upon. 
"\ATiere abscess occurs the operator is to blame. It means that pus- 
])roducing germs have been carried in upon the needle and that either 
the needle or the skin was not disinfected. Abscess is more likely to 
occur in horses and mules than in other animals. 

It has been the practice in Delaware to continue the vaccinated 
animals at their usual work. We have no data upon the subject show- 
ing this is unwise, but it is believed that the animals should be shielded 
as much as possible from excessive work and from extremes of heat 
or cold or from chilling rains. We advise our vaccinators, who con- 
sist of regular, practicing veterinarians designated by the governor 
upon the recommendation of the board of agriculture, to destroy all 
opened bottles of vaccine that remain unused at the end of a day's 
Vork, as it will certainly become contaminated and be spoiled if kept 
overnight at ordinary temperatures. The vaccine is dispensed in 
50-dose bottles hermetically sealed and distinctively labeled, so there 
can be no mistake made in using it. 

effecti\t:ness of the method. 

The results of vaccination have been as good, as shown by our sta- 
tistics, as those obtained by the use of any other biological product. 
That the method is entirely safe is shown by the fact that only one 
dangerous swelling has been brought to our notice in three years' 
experience in Delaware. Every year there are instances where, for 



26 ANTHRAX. 

some reason, animals that have been vaccinated die from anthrax, 
showing that they were not protected by the vaccine. No doubt some 
of these failures may be due to the animal being missed during vacci- 
nating. In other cases the failure may be due to the vaccine needle 
not properly puncturing the skin, so that the vaccine falls upon the 
ground. These accidents are readily brought about when the animals 
are unruly. On the other hand, the writer has observed a case of 
anthrax that occurred in vaccinated animals — ^that is, where it was 
positively certain that the vaccine was properly prepared and active 
and had been properly applied. 

Some cases of nonprotection by vaccination can be explained by 
the fact that the vaccine itself was inert, and therefore nonprotec- 
tive. Cultures made from such vaccine failed to grow. The vaccine 
would, of course, fail in the usual physiological test for an anthrax 
vaccine, and, of course, would fail to protect the animal against 
anthrax. The writer has known of instances where for various rea- 
sons an animal would escape vaccination, and that animal would be 
the only one to die on that farm. Hence he feels warranted in highly 
commending vaccination as the most important means of combating 
this terrible scourge. In France, where vaccination is most popular, 
and where statistics are reliable because of governmental control, the 
death rate before vaccination was adopted was, in cattle 5 per cent, 
and in sheep 10 per cent. After vaccination was adopted the losses 
were reduced to 0.34 per cent in cattle and 0.94 per cent in sheep. In 
Delaware the losses in 1907 in horses and cattle that were vaccinated 
were 0.32 per cent. 

Vaccination should be practiced every spring, at least a month be- 
fore it is time to turn the animals out to pasture, as a month is re- 
quired for the production of immunity. This vaccinating should not 
be optional with the owner, as at present, but should be compulsory, 
the State assuming the risk of loss from the use of the vaccine, but 
not in those cases where it can be shown the animal died not from 
vaccination but from a natural infection, owing to failure of being 
protected by the vaccine, or from other causes. 

A TEST OF PASTEUR VACCINES. 

Along with the investigation of anthrax for the purpose of dis- 
covering new methods of treatment and prevention of the disease, 
and for studying the biology of the causative organism and the gen- 
eral sanitary aspects of the subject, it was decided to test the efficacy 
of freshly prepared Pasteur vaccines. It is believed that some of the 
bad results that have been reported as following the use of Pasteur 
vaccine is due to carelessness on the part of those preparing and using 
it. As there is no visible difference in the appearance of the two 



METHODS OF PRODUCING IMMUNITY. 27 

vaccines, Nos. 1 and 2, it is an easy matter to get the bottles mixed 
or improperly labeled. If the labels should come off the bottles, the 
vaccinator would have no guide as to which vaccine he was using. 
The writer has purchased anthrax vaccines on the open market and 
has found some that were wholly inert, and others that were too 
strong when tested in the usual way. 

For the purpose of gaining such information as is possible from 
the practical experiences of practicing veterinarians who vaccinate 
against anthrax in Delaware, the writer undertook to prepare Pasteur 
vaccines for the State board of agriculture, which were to be used as 
soon as possible after they had reached destination. The cultures con- 
stituting the vaccines were grown in 50-dose bottles, each bottle con- 
taining 50 c. c. of bouillon which had been inoculated 24 hours pre- 
viously with the vaccinal germs. The official vaccinator was fur- 
nished first with No. 1 vaccine. In 12 days he was shipped the No. 2 
vaccine, for use on the same animals. Under this plan it was impos- 
sible for the bottles of vaccine to get mixed, even though the labels 
came off. 

The plan of using fresh vaccine in which the germ is still in the 
bacillar stage was continued with good results for two seasons. For 
the last two years the plan of using vaccines that had been incubated 
for four or five days, or until the bacilli had spored, has been tried. 
This latter method represents the condition of vaccine as put on the 
market by commercial houses, and is a suspension of spores instead 
of bacilli. According to the experiences of these two seasons, we 
have reason to believe that the vaccine will remain active for several 
months, and such cultures may be prepared several months in ad- 
vance of their use, provided the incubation is carried to a point 
where all growth ceases, or spores form. These spores inherit just 
that degree of strength possessed by their progenitors, and do not 
change except with a considerable lapse of time, possibly a year, if 
kept under favorable conditions. It is highly important that anae- 
robic conditions be not established in the bottles, as the bacilli will sink 
to the bottom and will not spore when grown anaerobically. To pre- 
vent this, it is important that the culture medium be made to reabsorb 
the air which has been driven out of it during sterilization before being 
inoculated with the vaccine germs. In preserving stock cultures of 
the vaccine it is very important that they be incubated for several 
days before being taken out of the incubator. If they are removed 
before spores have formed, the more vulnerable bacilli may succumb 
to existing unfavorable conditions. To these conditions may be 
ascribed the death of cultures of anthrax vaccine, and the loss of 
virulence of former virulent cultures of anthrax bacilli, which some- 
times occur during the course of laboratory work. 



28 ^^^^^^^^^ ANTHRAX. ^^^^^^^^^^^^^™ 

EXPERIMENTS WITH VARIOUS SUBSTANCES TO TEST IMMUNIZING 

POWERS. 

Experiments upon the disease have been carried on uninterruptedly 
for the past three years. These had for their object the preparation 
of substances in the laboratory and in living animals which could 
be used in combating anthrax by acting as antitoxins or bactericides, 
or as vaccines. 

Realizing the great importance and economy in producing pro- 
tective and curative substances in the laboratory over the necessarily 
expensive methods when employing animals for the same purposes, 
efforts have been made along the line of producing various culture 
products to be used in combating anthrax. 

Pyocyanase, made after the method of Emmerich and Loew, gave 
some good results when tested upon rabbits, but failed upon sheep. 

Anthraxase, prepared by the writer after the same general method 
used in producing pyocyanase, was without protective or curative 
properties, although it produced high fever when injected into rabbits 
and sheep subcutaneous^. 

Anthraxoin, consisting of a turbid suspension of dead anthrax 
bacilli, was apparently useless in protecting sheep against anthrax 
when used similarly to the Pasteur vaccine. 

A single vaccine, having for its object the production of immunity 
in two weeks and thus cutting down the necessary period by one-half, 
was made by incubating a virulent bacillus for about 18 days at 42° 
to 43° C. Such a culture will kill guinea pigs in about a week, and 
in strength it thus holds a position between the two vaccines of 
Pasteur. With it sheep were vaccinated and after 12 days withstood 
an otherwise fatal infection with virulent bacilli. In some cases, 
however, the immunity was not sufficiently strong, as was evidenced 
by the death, now and then, of a sheep when tested with virulent 
bacilli. 

The preparation and use of the various substances, together with 
the results of the experimental work, are described in the following 
pages. 

ANTHRAXIN. 

Anthraxin was made similarly to tuberculin and mallein. The 
cultures of anthrax were grown for 10 days with daily shaking in 
glycerinated, peptonized bouillon, the glycerin being used in 4 per 
cent strength. The cultures were then sterilized by boiling, filtered 
through Berkefeld filters, and then evaporated to one-tenth of the 
original volume. A sirupy liquid, much resembling tuberculin, re- 
sulted. It was tested on rabbits and sheep for immunizing proper- 
ties, as shown in Table 1, on the following page. 



EXPERIMENTS' WITH ANTHRAXIN. 



29 



Table 1. — Experiments with anthraxin. 





Date. 


Injections. 


Temperature. 




Animal. 


Anthraxin. 


Virulent 
culture 
anthrax. 


Result and remarks. 


Rabbit 1 


1907. 
July 10 


1 c. c. subcutane- 
ous. 




°F. 






July 17, 10 a. m. . . 




105, 3 p. m 

104, 10a. m... 

103, 3 p. m 

Nonnal 

104 

105 






July 18 










do 










July 19 










July 24, 11 a. m. . . 
July 24, 1.30 p. m.. 


1 c. c. subcutane- 
ous. 














July 24, 5.30 p.m.. 






105 






July 25, 9 a. m 






Normal 






July 31, 9 a. m 




0.05C.C. 






Aug. 2, 9 a. m 




Dead from anthrax. 


Sheep 1 


Aug. 27, 1 p. m — 
Aug. 27. 6 D. m 


5 c. c. subcutane- 
ous. 












Sheep blowing, head 

do\vn. 
Sheep seems well. 




Aug. 28, 9 a. m 










Sept. 3, 9 a. m 

Sept. 10, 9 a. m.. . 


5 c. c. subcutane- 
ous. 








0. 25 c. c. 








Sept. 12, 9 a. m 




Dead from anthrax. 


Sheep 2 


Aug. 27, 1 p. m 

Aug. 27, 6 p. m 


5 c. c. subcutane- 
ous. 












Sheep blowing, head 

down. 
Seems well. 




Aug. 28, 9 a. m 










Sept. 3,9 a. m 

Sept. 10,9 a. m 


5 c. 0. subcutane- 
ous. 
do 


















Sept. 23,9 a. m 




0. 25 c. c. 








Sept. 25, 9 a. m 




Dead from anthrax. 


Sheep 3 


Aug. 27, 9 a. m 

Aug. 27, 6 p. m... 


5 c. c. subcutane- 
ous. 














Blowing; no fever. 




Sept. 3, 9 a. m 

Sept. 5 


5 c. c. subcutane- 
ous. 












Diarrhea. - 




Sept. 10, 9a. m... 
Sept. 15, 9 a. m. . . 


5 c. c. subcutane- 
ous. 






Well. 




0. 25 c. c. 








Sept. 17, 9 a. m. . . 




Dead from anthrax. 


Sheep 4 


Aug. 27, 9 a. m 

Aug. 11, 6p. m 


5 c. c. subcutane- 
ous. 












Blowing; no fever. 




Sept. 13, 9 a. m... 

Sept. 10, 9 a. m.. . 
Sept. 15, 9 a. m. . . 


5 c. c. subcutane- 
ous. 
do 






Diarrhea for several 








days. 






0. 25 c. c. 








Sept. 17, 9 a. m. . . 




Dead from anthrax. 


Sheep 5 and 6. . . 


Sept. 3, 9 a. m 

Sept. 6 


5 c. c. subcutane- 
ous. 














Seemed well. 




Sept. 10, 9 a. m... 
Oct. 7, 9 a. m 


5 c. c. subcutane- 
ous. 










0. 25 c. c. 








Oct. 9, 9 a. m 




Both dead from an 












thrax. 



From the foregoing experiments we gather that anthraxin was 
possessed of no immunizing properties whatever. 



30 



ANTHRAX. 



PYOCYANASE. 



Pyocyanase, the next substance experimented with, was prepared 
as follows: Large flasks of medium were inoculated with Bacillus 
fyocyaneus and grown at 37° C. until a ropy condition was produced, 
which required three weeks, the flasks meanwhile being shaken daily. 
The composition of the medium was as follows: Peptone, 0.5 per 
cent; glycerin, 0.1 per cent; dipotassium phosphate, 0.1 per cent; 
magnesium sulphate, 0.01 per cent; sodium chlorid, 0.3 per cent; 
sodium bicarbonate, 0.1 per cent; in distilled water (synthetic medium 
of Emmerich and Loew). When growth had ceased in this medium 
the growths from a number of agar-agar cultures of the same organ- 
ism were added and the whole was thoroughly shaken. The culture 
medium was then neutralized with dilute hydrochloric acid. Car- 
bolic acid was then added to 0.2 per cent strength as a preservative. 
The liquid was then evaporated down to one-tenth of its original 
volume at ordinary room temperature by being placed in pie plates. 
Then the liquid was dialyzed for 24 hours in running water, filtered 
through Berkefeld filters, and 0.2 per cent carbolic acid was again 
added to replace that which was dialyzed out. The resulting liquid, 
pyocyanase, has a dark coffee color and a pungent odor. The experi- 
ments to test its immunizing and curative properties upon guinea 
pigs, rabbits, and sheep are recorded in Tables 2 and 3. 

Table 2. — Bxper'iments tcith unfiltered pyocyanase. 





Date. 


Injections. 


Temper- 
ature. 




AnimaL 


Pyocyanase. 


Virulent 24- 
hour culture 
anthrax. 


Result and remarks. 


Eabbit 1 


1907. 
Dec. 9, 9 a. m 

Dec. 10, 9 a. m 

Dec. 11, 9 a. m 


6 c. c. sub- 
cutaneous. 

4 c. c. sub- 
cutaneous. 




° F. 






0.2 c.c. sub- 
cutaneous. 


















Rabbit dead. Liver ne- 


Guinea pig 1 




3 c. c. sub- 
cutaneous. 

1\ c. c. sub- 
cutaneous. 

2 c. c. sub- 
cutaneous. 

3 c. c. sub- 
cutaneous. 






crosed, spleen enlarged; 
anthrax bacilli at local 
lesion, but none found in 
blood or in liver and 
spleen; probably died from 
pyocyanase poisoning. 




Dec. 17, 10 a. m... 
Dec. 18, 10 a. m... 
Dec. 18, 1.30 p.m.. 

Dec. 16, 10 a.m... 
Dec. 17, 10 a.m... 


0.1 c. c. sub- 
cutaneous. 


















Died during night. No ba- 


Guinea pig 2 






cilli in blood, but few at 
local lesion; probably died 
from pyocyanase poison- 
ing. 


0.1 c. c. sub- 
cutaneous. 













EXPERIMENTS WITH UNFILTEKED PYOCYANASE. 31 

Table 2. — Experiments with unfiltered pyocyanase — Continued. 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Pyocyanase. 


Virulent 24- 
hour culture 
anthrax. 


Result and remarks. 


Guinea pig 2 . . . 


1907. 
Dec. 18, 9 a. m 

Dec. 19, 9 a. m 


li c. c. sub- 
cutaneous. 




" J^. 


Sick. 






Chloroformed. No bacilli in 


Guinea pig 3.... 


Dec. 19, 9 a. m 

Dec. 20, 10a.m... 
Dec. 21, 3p. m 

1908. 
Jan. 2, 9 a. m 


3 c. e. sub- 
cutaneous. 
do 






blood, but a few at local 
lesion ; pyocyanase poi- 
soning. 










1| c. c. sub- 
cutaneous. 








0. Ice. sub- 
cutaneous. 








Jan. 4 9 a. m 






Dead from anthrax. Bacilli 


Rabbit 2 


Jan. 13 9 a. m 

Jan. 14, 9 a. m 

Jan. 15, 9 a. m 


6 c. c. sub- 
cutaneous. 
do 


0.2 c.c. sub- 
cutaneous. 




in blood; another giiinea 
pig treated similarly died 
of anthrax on third day 
after inoculation. 




103 . 
102 












Jan. 16, 9 a. m. 






Found dead of anthrax. 


Rabbits 


Jan. 13, 9 a. m 

Jan. 14, 9 a. m 

Jan. 15, 9 a. m 

Jan. 18, 9 a. m 


6 c. c. sub- 
cutaneous. 

6 c. c. sub- 
cutaneous. 






Bacilli in blood. 




0.3 c. c. sub- 
cutaneous. 




Edema at points of injection 




Normal. 


of pyocyanase; no fever. 






Animal apparently well. 




Jan. 25, 9 a. lu 








Animal dies from anthrax 


Rabbit 4 


Jan. 13 9 a. m 

Jan. 14.9 a. m 

Jan. 15, 9 a. m 


6 c. c. sub- 
cutaneous. 

do 

do 


0.2 c. c. sub- 
cutaneous. 




on eleventh day. Bacilli 
in blood. 




Normal, 
do.... 






Jan. 16, 9 a. m 








Dead from anthrax. An- 


Rabbits 


Feb. 3, 10 a. m 

Feb. 4, 10 a. m 

Feb. 5, 10 a. m 


J c. c. sub- 
cutaneous. 
do 

. do.... 


- 




other rabbit treated simi- 
larly died of anthrax on 
third day also; bacilli in 
blood. 




0.3 c. c. sub- 
cutaneous. 


102.8 


Slight swelling at point 
where pyocyanase was in- 
jected. 




Feb. 8, 10 a. m 








Animal dead from anthrax, 


Rabbit 6 


Feb. 3, 10 a. m 

Feb. 4, 10 a. m 

Feb. 5 10 a. m 


Ice. sub- 
cutaneous. 
do 

do.... 






having lived four days. 




0.3 c. c. sub- 
cutaneous. 




Considerable reaction at 






point of injections. 




Feb. 7, 10 a. m 








Rabbit dead from anthrax 


Rabbit 7 


Feb. 3, 10 a. m 

Feb. 4, 10 a. m 


2 c. c. sub- 
cutaneous. 
do- 






on third day. 




0.3c.c. sub- 
cutaneoTis. 


103 
103 


Small reaction from injec- 




Feb. 5, 10 a. m 




tions. 




Feb. 8, 10 a. m 






Animal dead from anthrax, 












having lived four days. 



32 ANTHRAX. 

Table 2. — Experiments tvith unfiltered pyocyanase — Continued. 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Pyocyanase. 


Virulent 24- 
hour culture 
anthrax. 


Result and remarks. 


Rabbits 


1908. 
Feb. 3, 10 a. m 

Feb. 4, 10 a, m 

Feb. 7, 10 a. m 


4 c. c. sub- 
cutaneous. 

1 c. c. sub- 
cutaneous. 




°F. 






0.3 c.c. sub- 
cutaneous. 


103.5 


Some reaction from injec- 
tions. Abscess formed at 
point of injection. 

Animal dead from anthrax. 


Rabbit 9 


Feb. 3, 10 a. m 

Feb. 4, 10 a. m 

Feb. 5, 10 a. m 


5 c. c. sub- 
cutaneous. 

2 c. c. sub- 
cutaneous. 










0.2 c. c. sub- 
cutaneous. 




Severe reaction at point of 

injection. 
Abscess forms. 




104 
105 




Feb. 8, 10 a. m 






Animal dead from anthrax 










on fourth day. 



The foregoing experiments were made with unfiltered pyocyanase. 
The dead bacilli produce abscesses at points of injection and fre- 
quently disease of the liver, which contributed to death from anthrax. 

Table 3. — Experiments loith filtered pyocycbnase. 





Date. 


Injections. 


Tem- 
perature. 




Animal. 


Pyocyanase. 


Virulent 24- 
hour culture 
of anthrax. 


Result and remarks. 

• 


Sheep 1 


1909. 
Jan. 4, 10 a. m 

Jan. 4, 1 p. m 


10 c. c. sub- 
cutaneous. 




°F. 
103.6 

105 
106 

103.6 
103 












Jan. 4, 3p. m 

Jan. 5, 9 a. m 


10 c. c. sub- 
cutaneous. 


0.1 c. c. sub- 
cutaneous. 


Animal sick. 




Jan. 5, 4 p. m 






Ofl feed. 




Jan. 5, 7p. m 






Dead from pyocyanase poi- 
soning. 


Sheep 2 


Jan. 6, 9 a. m 

Jan. 6, 12 m 


3 c. c. sub- 
cutaneous. 




103 

105. 5 
106.9 
104 










Jan. 6, 4 p. m 










Jan. 7, 10 a. m 

Jan. 7, 11 a. m 


1 c. c. sub- 
cutaneous. 










Respirations jerky. 




Jan. 21, 3p.m 

Jan. 22, 9 a. m 

Jan. 22, 3 p. m 

Jan. 23, 9 a. m 

Jan. 23, 3 p. m 

Jan. 25, 9 a. m 


5 c. c. sub- 
cutaneous. 

4 c. c. sub- 
cutaneous. 

2 c. c. sub- 
cutaneous. 

3 c. c. sub- 
cutaneous. 

2 c. c. sub- 
cutaneous. 










103.5 

102.5 

102.5 

103 

102 
102 


















0.1 c. c. sub- 
cutaneous. 






Jan. 26, 12 m 










Jan. 28, 10 a. m 






Dead from anthrax, having 


Sheep 3 


Jan. 7, 10 a. m 

Jan. 7, 4p. m 


2 c. c. sub- 
cutaneous. 




102 
107.2 

101 
101 


lived 5 days. 






Animal does not seem sick 




Jan. 13, 9 a. m 

Jan. 13, 3p. m 


2 c. c. sub- 
cutaneous. 

5 c. c. sub- 
cutaneous. 




even though temperature 
is high. 




0.1 c. c. sub- 
cutaneous. 





EXPERIMENTS WITH FILTERED PYOCYANASE. 
Table 3. — Experiinents ivith filtered pyocyamase — Continued. 



33 





Date. 


Injections. 


Tem- 
perature. 




Animal. 


Pyocyanase. 


Vinilent 24- 
hour culture 
of anthrax. 


Result and remarks. 


Sheep 3 


1909. 
Jan. 14, 9 a. m 

Jan. 14 1.30 p. m.. 


2 e. 0. sub- 
cutaneous. 




"F. 
102.5 

102.5 
104.5 












Jan. 15, 10 a. ni — 
Jan. 16 


2 c. c. sub- 
cutaneous. 




Anthrax fever setting in. 
Dead from anthrax (3 days;. 






Sheep 4 


Jan. 8, 10 a. m 

Jan. 8, 1 p. m 


1 c. c. sub- 
cutaneous. 




101 
102 








Jan. 8, 3p. m 

Jan. 9, 10 a. m 


1 c. c. sub- 
cutaneous. 
do 


0.1 c. c. sub- 
cutaneous. 






103 

103.5 

105.5 

103.3 
103 






Jan. 10, 4.30 p. m.. 
Jan. 11, 9 a. m 


do 




Dead two hours later of an 


Rabbit 10 


1908 
Mar. 5, 9 a. m 

Mar. 6, 9 a. m 

Mar. 7, 9 a. m 


0.05 e.c. sub- 
cutaneous. 

0.5 c. c. sub- 
cutaneous. 
do 




thrax (3 days). 
















Mar. 8, 9 a. m 

Mar. 9, 9 a. m 

Mar. 13, 9 a. m 


0.5 c. c. sub- 
cutaneous. 


0.2 e. c. sub- 
cutaneous. 








102.8 


Rabbit well. 










Mar. 18, 9 a. m 








Found dead of anthrax (10 


Rabbit 11 


Mar. 5, 9 a. m 

Mar. 6, 9 a. m 


1 c. c. subcu- 
taneous. 
do 






days). 






103 






Mar. 7, 9 a. m 

Mar. 8, 9 a. m 


do 


0.2 c. c. sub- 
cutaneous. 






103 

101.5 

102.6 

103.7 

103 

103 






Mar. 10, 9 a. m 










Mar. 18, 9 a. m 

Mar. 21, 9 a. m 

Mar. 23, 9 a. m 

Mar. 24, 9 a. m 


1.5 c. c. sub- 
cutaneous. 

3 c. c. sub- 
cutaneous. 

5 c. c. sub- 
cutaneous. 
















Animal sick. 
. Animal much better. 




Apr. 12, 9 a. m 






Much emaciated and swol- 




Apr. 13, 9 a. m 








len along belly. 
Dead from anttirax. Post- 


Rabbit 12 


Mar. 5, 9 a. m 

Mar. 6, 9 a. m 


1.5 c. c. sub- 
cutaneous. 
do 




102 

102 
103 

102.8 
102.8 
103 

104.3 


mortem e x a m i n a tion 
showed linear abscess in 
Uver; repeated larger doses 
of pyocyanase probably 
caused animal to succumb 
to anthrax (lived 5 weeks); 
check rabbit died in 48 
hours after inoculation. 










Mar. 7, 9 a. m 

Mar. 8, 9 a. m 


do 

do 


0.2 c. c. sub- 
cutaneous. 






Mar. 18, 9 a. m 

Mar. 20, 9 a. m 

Mar. 24, 9 a. m 

Apr. 12, 9 a. m 


do 








3 c. c. sub- 
cutaneous. 

5 c. c. sub- 
cutaneous. 










Animal has kept well. 




0.1c. c. sub- 
cutaneous. 






Apr. 14, 9 a. m 






Animal found dead from 


Rabbit 13 


Mar. 18, 9 a. m 

Mar. 18, 2 p. m 


5 c. c. sub- 
cutaneous. 
do 






anthrax, showing immu- 
nity was only passive 
(lived 24 days). 




0.2 c. c. sub- 
cutaneous. 













34 ANTHRAX. 

Table 3. — Experiments with filtered pyocyanase — Continued. 





Date. 


Injections. 


Tem- 
perature. 




Animal. 


Pyocyanase. 


Virulent 24- 
hour culture 
of anthrax. 


Result and remarks. 


Kabbit 13 


1908. 
Mar. 18, 4 p. m 






°F. 
104 
104 
101 






Mar. 19, 9 a. m 










Mar. 20, 9 a. m 

Mar. 28 


5 c. c. sub- 
cutaneous. 










Rabbit found dead from an- 


Rabbit 14 


Nov. 24, 9 a. m 

Nov. 24, 3 p. m . . . 
Dec. 4, 9 a. m 


1 c. c. sub- 
cutaneous. 
do 

do 






thrax. Period of incuba- 
tion lengthened to 10 days 
by pyocyanase; two more 
rabbits similarly treated 
reacted the same, and 
lived 10 days and 7 days, 
respectively. 

Simultaneous injections of 




0.2 c. c. sub- 
cutaneous. 


103 


"ase" and bacilli. 




Dec. 5 












Dec. 16 


3 c. c. sub- 
cutaneous. 










Feb. 4 






Found dead from anthrax. 


Rabbit 15 


Nov. 24, 11 a. m. . . 
Nov. 24, 3. p m . . - 


2 c. c. sub- 
cutaneous. 
do 






Lived 21 days. 




0.2 c. c. sub- 
cutaneous. 


104 






Dec. 4, 9 a. m 


do 


Some swelling which disap- 




Dec.5.. 








pears in 2 days. 
Animal has remained well 




Dec. 16 


3 c. c. sub- 
cutaneous. 






up to Feb. 24, when it was 




Feb. 4 






killed by dog (lived 69 
days). 


Rabbit 16 


Nov. 24, 11a. m... 
Nov. 24,3 p.m.... 
Dec. 4, 9 a. m 


3 c. c. sub- 
cutaneous. 
do 

do 










0.2 c. c. sub- 
cutaneous. 


104 


Animal remains well. 




Dec. 5, 9 a. m 






104.8 


Swollen from "ase" injec- 
tion. 




Dec. 16, 9 a. m 

Feb. 4 


3 c. c. sub- 
cutaneous. 










Animal has remained well 


Rabbit 17 


Nov. 24, 11 a. m. . . 
Nov. 24, 3 p.m.... 


4 c. c. sub- 
cutaneous. 
do 






up to Feb. 4, when it was 
killed by dog (lived 69 
days). No lesions on post- 
mortem; check rabbit died 
in 48 hours after inocula- 
tion. 

Simultaneous injection. 




0.2 c. c. sub- 
cutaneous. 


104 


Chloroformed to locate posi- 
tion and stage of develop- 
ment of bacilli. No spores 
or bacilli found by micro- 
scope or culture in local 
lesion, liver, spleen, or 
peritoneal cavity. Check 
rabbit died in 48 hours 
after inoculation. 



From the foregoing experiments with pyocyanase it will be seen 
that when given in proper dose and simultaneously with virulent 
anthrax bacilli, the period of inoculation is greatly lengthened in 
rabbits. Sheep, however, seem to be very susceptible to poisoning by 
pyocyanase, and no immunity is conferred upon them by its use. 



EXPERIMENTS WITH ANTHEAXASE. 



35 



ANTHRAXASE. 

Anthraxase was made in about the same manner as pyocyanase, 
except that the Bacillus anthracis was substituted for Bacillus pyo- 
cyaneus. The tests of this substance include cultures grown in ordi- 
nary bouillon, cultures grown in Emmerich and Loew's medium, and 
experiments with precipitated anthraxase. The details are given in 
Tables 4, 5, and 6. 



Table 4. — Experiments ivith anthraxase — Cultures grown in ordinary touillon. 









Injections. 


Temper- 
ature. 




Animal. 


Date. 


Anthraxase. 


Anthrax 
cultmre. 


Results and remarks. 


Rabbit 18 


1908. 
Mar. 17- Q a- m 


0.75 c.c. sub- 
cutaneous. 

0.50 c.c. sub- 
cutaneous. 
do 

3 c. c. subcu- 
taneous. 

5 c. c. subcu- 
taneous. 

do 




101 

101.5 

102 

102.4 

107 






Mar. 18 

Mar. 19 

Mar. 20 

Mar. 21 

Mar. 23 
Mar. 25 

Mar. 17 

Mar. 18 
Mar. 19 

Mar. 20 

Mar. 21 

Mar. 17 

Mar. 18 

Mar. 19, 

Mar. 20 
Mar. 21 

Mar. 22 

Mar. 23 
Mar. 24, 


9 a. m 

9 a. m 

9 a. m 

9 a. m 

9 a. m 








0. 2 c. c. sub- 
cutaneous. 








Anthrax fever. 






Animal very sick. 
Dead from anthrax (lived 
5 days). 




9 a. m 








Rabbit 19 


9 a. m 

9 a. m 


1.5c.c.sub- 
cutaneous. 
. ...do. 




100.5 

102 
102.6 

104.6 






Swollen lymphatics. 
Anthrax fever. 




9 a. m 

9 a. m 

9 a. m 


2 c.c. subcu- 
taneous. 

3 e. c. subcu- 
taneous. 


0.2 c. c. sub- 
cutaneous. 






Dead from anthrax on sec- 


Rabbit 20 


9 a. m 

9 a. m 


1 c. c. subcu- 
taneous. 
do 




101 
101.5 


ond day. 










9 a. m 

9 a. m 




0.2 c. c. sub- 
cutaneous. 






104.1 
103 


Anthrax fever. 




9 a. m 

9 a. m 


3 c.c. subcu- 
taneous. 










Dead from anthrax on third 


Rabbit 21 . . 


9 a. m 

9 a. m 


0.5 c. c. sub- 
cutaneous. 
do 




103 

104 
104 


day. 
Animal pregnant. 


















Mar. 2G, 


9 a. m 


5 c. c. intra- 
venously. 

3 c. c. subcu- 
taneous. 

2 c. c. subcu- 
taneous. 
do 








do 

Mar. 23, 9 a. m 


0.2 c. c sub- 
cutaneous. 




Animal died from shock. 


Rabbit 22 


104.5 

104 
103.1 






Mar. 24 
Mar. 26, 

do. 


9 a. m 








9 a. m 


5 c. c. intra- 
venously. 

3 c.c. subcu- 
taneous. 


0.2 c. c. sub- 
cutaneous. 






Mar. 27 
Mar. 28 

Mar. 30 

Mar. 23 

Mar. 24 
Mar. 25 
Mar. 26, 


9 a. m 




102.9 
102.4 






9 a. m 

9 a. m 


4 c. c. subcu- 
taneous. 










Died of anthrax on fourth 


Rabbit 23 


9 a. m 

9 a. m 


4c. 0. subcu- 
taneous. 
do 




105 
106.5 


day. 
Animal excited. 










9 a. m 










9a. ni 


5 c. c. subcu- 
taneous. 


0.2 c. c. sub- 
cutaneous. 


103.4 





36 ANTHRAX. 

Table 4. — Experiments with anthracease, etc.- — Continued. 



* 


Date. 


Injections. 


Temper- 
ature. 




Animal. 


Anthraxase. 


Anthrax 
cultm-e. 


Results and remarks. 


Rabbit 23 


1908. 
Mar. 28. 9 a. m 

Mar. 30, 9 a. m 


4c. c. subcu- 
taneous. 




°F. 










Died of anthrax on third 


Rabbit 24 


Mar. 23, 9 a. m 

Mar. 24, 9 a. m 


6 c. c. subcu- 
taneous. 
do 




104.1 

105 
103.9 


day. 
Animal pregnant. 








Mar. 25, 9 a. m 










Mar. 26, 9 a. m 

do 

Mar. 28,9 a. m 

Mar. 30, 9 a. m. .. 


5 c. c. intra- 
venously. 

3 cc. subcu- 

taneous. 

4 c. c. subcu- 

taneous. 








0.2 c. c. sub- 
cutaneous. 


103.6 








Dead from anthrax on fourth 


Rabbit 25 


Mar. 25,9 a. m 

Mar, 26, 9 a. m 

do 


3 c. c. intra- 
venously. 

5 c. c. intra- 
venously. 

3 c. c. subcu- 
taneous. 

0.5 c. c. sub- 
cutaneous. 
do 


0.1 c. c. sub- 
cutaneous. 


103 
103.8 


day. 
Animal pregnant. 

Much depressed by the in- 
jections and aborted at 4 
p. m. (Control for this se- 
ries dies in 48 hours after in- 
oculation with the same 
culture.) 






Rabbit 26 


Apr. 7,9a.m..... 
Apr. 8, 9 a. m 












102 
103 






Apr. 9, 9 a. m 

AiDr. 10, 9 a. m 


do 

do 


0.1 c. c. sub- 
cutaneous. 






Apr. 11, 9 a. m 

Apr. 11, 4 p. m 

Apr. 12, 9 a. m 


2 cc. subcu- 
taneous. 

3 c. c subcu- 

taneous. 




105 
105.3 














Dead from anthrax on third 












day. 



Table 5. — Experiments toith anthraxase. Cultures groion in Emmerich and 

Loew's medium. 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Anthraxase. 


Anthrax cul- 
ture. 


Results and remarks. 


Rabbit 27 


1908. 
Apr. 7, 10 a. m 

Apr. 9, 10 a. m 

Apr. 10, 10 a. m. . . 
Apr. 11, 10 a. m... 

Apr. 11, 4 p. m 

Apr. 12, 4 p. m 


0.5 c c. sub- 
cutaneous. 
do 

do 

1 0. c. sub- 
cutaneous. 
3 c c. sub- 
cutaneous. 




°F. 
102 

102 

102 
102.3 

102.5 

103 






0.2 c. c. sub- 
cutaneous. 














Much swollen. 




Apr. 13, 4 p. m 






Dead from anthrax on fifth 


Rabbit 28 . 


Apr. 15, 10a. m... 
Apr. 16, 10a. m... 


0.25 c c sub- 
cutaneous. 
do 






day. 






100.8 
99.5 
99.8 

101 

102.8 






Apr. 17, It) a. na. . 


do 








Apr. 18, 10a. m... 


do 








Apr. 25,10a. m... 
Apr. 26,10a.m... 
Apr. 27, 10 a. m.. 


do 

0.50 c c sub- 
cutaneous. 


0.2 c c sub- 
cutaneous. 








Dies of anthrax in 2 days. 













EXPERIMENTS WITH ANTHRAXASE. 



37 



The following experiments on rabbits and sheep were made with 
precipitated anthraxase. Absolute alcohol, 9 volumes to 1 of anthrax- 
ase solution, was employed as the precipitant. The resulting yellow, 
gummy precipitate was readily soluble in water, to which 0.2 per cent 
strength of carbolic acid was added. 

Table 6. — Experiments tcith precipitated anthraxase. 



I 



I 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Anthraxase. 


Anthrax 
culture. 


Result and remarks. 


Rabbit ''o 


190S. 
Apr. 15, 9 a. m 

Apr. 16,9 a. m 


0.5 c, c. sub- 
cutaneous, 
.do 




°F. 
101 

101 
101 
101 












Apr. 17,9 a. m 


.do 








Apr. 18, 9 a ra 


do 








Apr. 25, 9 a. m 

Apr. 26, 9 a. m 

Apr. 27,9 a. m 

Apr. 27, 5 p. m 

Apr. 28-9 a. m 


do 

do 

do 

5 c. c. sub- 
cutaneous. 


0.01 c.c. sub- 
cutaneous. 






101.8 
102 


Check rabbit dies. 






103 
103 






Apr. 29,9 a. m. .. 










Apr. 30 






Animal seems perfectly 

well. 
Given to test duration of 




Nov. 5, 9 a. ra. 




0.1c. c. sub- 
cutaneous. 






Nov. 9.9 a. m 






immunity. 
Found dead of anthrax. 


Sabbit 30 


May 2, 10 a. m 

May 2, 2 p. m 


1 c. c. sub- 
cutaneous. 
do 




103.3 

102.5 
103 

102 

104.5 
105 

104 
100.3 

102. 3 

301 
102 

104.8 

102. 5 

101 
102 
102.5 












May 2, 4p. m 

May 4, 10 a. m 

May 4, 4 p. m 


do 

3 c, c. sub- 
cutaneous. 


0.1 c. c. sub- 
cutaneous. 












Mays, 11.30 a. m.. 
May 5, 4 p. m . . . 


2 c. c. sub- 
cutaneous. 










' 




May 6, 10 a. m 






Dead of anthrax at 4 p. m. 
on fifth day. 


Rabbit 31 


May 2, 10 a. m 

May 2, 2 p. m 


1 c. c. sub- 
cutaneous. 
do 












May 2, 4p, m 

May 4, 10 a. m 


do 


0.1 c. c. sub- 
cutaneous. 


Anthrax fever. Check dies 




May 5, 10 a. m 

May 5, 4 p. m 


2 c. c. sub- 
cutaneous. 




to-day. 










May 6, 10 a. m 










May 7, 10 a. m 










May 8, 10 a. m 






Animal turned out in yard. 
Animal found dead. Post- 




May 14, 10 a. m . . . 








Sheep 5 


June 29, 10 a. m... 
June 29, 12 m 


5 c. c. sub- 
cutaneous. 
do 






mortem showed death 
from anthrax and cocci- 
diosis (lived 12 days). 








June 29, 2 p. m 


do 










June 29, 4 p. m 

June 30, 10 a. m ... 


do 

. ...do 


0.1c. c. sub- 
cutaneous. 














June 30,12 m 


do 










June 30, 2p. m 


do 










June 30, 4 p. m 


do 










July 2, 9 a. m 


do 




105. S 
104.3 
105.8 






July 2, 10 a. m 


do 








July 2, 12 m 


do 




Died of anthrax at 2 p.m. 
on fourth day. 


Sheep 6 


Jime 29, 10 a. m... 


. ...do 






June 29, 12 m 


do 










June 29, 2 p. m 


do 









38 ANTHRAX. 

Table 6. — Experiments with precipitated anthraxase — Continued. 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Anthraxase. 


Anthrax 
culture. 


Result and remarks. 


Sheep 6 


1908. 
June 29, 4p. m 

June 30, 10 a. m... 


5 0. c. sub- 
cutaneous. 
do 


0.1 c. c. sub- 
cutaneous. 


°F. 












June 30, 12 m 


do 










June 30, 2 p. m 

June 30, 4 p. m 


do 










do 










July 2, 8 a. m 


do 




102.2 

102 
102 
102.8 
103 


Check guinea pig dies in 2 
days. 




July 2, 10 a. m 


do 






July 2, 12 m 


do 








July 2, 3 p. m 


...do 








July 2, 7p. m 


do 








July 4, 10 a. m 


do 




Animal dead of anthrax on 


Sheep 7 


July 28, 10 a. m . . . 


.....do 






sixth day. 




July 28, 12 m 


do 










July 28, 2p.m 


do 










July 28, 4p. m 

July 29, 10 a. m . . . 


do 

do 


0.1c. c. sul- 
cutaaeous. 








102.3 

102 

102.3 

102.6 

106 

106 

106 

106.2 






July 29, 12 m 


do 








July 29, 2p. m 


do 








July 29, 4p. m 


do 








July 30, 10 a. m... 


do.. .. 








July 30, 12 m 


do. . 








July 30, 2p. m 


do 








July 30, 4p. m 

July 31, 10 a. m ... 


do 




Found dead of anthrax on 












fourth day after inocula- 
tion of test dose. 



ANTHRAXOIN. 

Anthraxoin was prepared and experimented with as follows: It 
consists essentially of a suspension, in carbolized normal salt solution, 
of dead, sporeless, anthrax bacilli. It was produced by inoculating, 
from a fresh anthrax carcass, bottles that had been filled completely 
with nutrient bouillon and plugged with hardest paraffin stoppers. 
In this way none but sporeless bacilli were introduced, and under the 
existing anaerobic conditions no spores could form. After all growth 
had ceased, at 35° C, these cultures were completely immersed in a 
water bath maintained at 55° C. for one hour, which was sufficient 
to devitalize the bacilli but not to destroy any antibodies they might 
contain. The dead bacilli were separated by filtration from the 
liquid in which they had grown, and those adhering to the Berkefeld 
filter were washed off in the requisite amount of carbolized normal 
salt solution. The suspension experimented with consisted of the 
bacilli that grew in 2,500 c. c. of bouillon, suspended in 50 c. c. of 
carbolized normal sodium chlorid solution. The details are given in 
Table 7. 



EXPEKIMENTS WITH ANTHRAXOIN. 
Table 7. — Experiments with anthraxoin. 



39 





Date. 


Injections. 


Temper- 
ature. 




Animal. 


Anthraxoin. 


Anthrax cul- 
ture. 


Result and remarks. 


Sheep 8. . . 


1908. 
Apr. 24, 10 a. m... 

May 8, 10 a. m 

May 20, 10 a. m... 


1 c. c. subcu- 
taneous. 

2 c. c. subcu- 
taneous. 




"F. 














0.25 c. c. sub- 
cutaneous. 








May 22,10 a. m.. . 






Sheep dies of anthrax in 2 
days. 


Sheep 9 


Apr. 24, 10a.m... 
May 8, 10 a. m 


2 c. c. subcu- 
taneous. 








0.25 c. c. sub- 
cutaneous. 








May 9, 10 a. m 




102.2 
103 
101.5 
101.5 






May 9, 4 p. m . 










May 10, 10 a. m. . . 










May 11,10 a. m. . . 






Remains well. 


Sheep 10 


Apr. 24, 10 a.m... 

Mays, 10 a. m 

May 20,10 a. m 


0.5 c. c. sub- 
cutaneous. 

1 c. c. subcu- 
taneous. 














0.25 c. c. sub- 
cutaneous. 




Sheep dies of anthrax in 3 
days. 


Sheep 11 


May 11, 10 a. m 

May 24, 10 a. m 


1 c. c. subcu- 
taneous. 




0.25 c. c. sub- 
cutaneoiis. 




Sheep dies of anthrax in 2 
days. 


Sheep 12 


May 11, 10 a. m 

May 20, 10 a. m 


1 c. c. subcu- 
taneous. 




0.25 c. c. sub- 
cutaneovis. 




Sheep dies of anthrax in 3 
days. 


Sheep 13 


May 11, 10 a. m 

May 23, 10 a. m 

Jime 8, 10 a. m 


1 c. c. subcu- 
taneous. 

5 c. c. subcu- 
taneous. 












0.25 c. c. sub- 
cutaneous. 








June 10, 10 a. m . . . 






Sheep dead from anthrax in 
2 days. 


Sheep 14 


May 11, 10 a. m 

May 23, 10 a. m 

June 8, 10 a. m 


1 c. c. subcu- 
taneovis. 

5 c. 0. subcu- 
taneous. 














0.25 c. c. sub- 
cutaneous. 








June 12, 10 a. m... 






Sheep dead from anthrax in 
4 days. 


Sheep 15 


Apr. 26, 9 a. m 

Apr. 26, 12 m 


10 c. c. sub- 
cutaneous. 
.. ..do 




103 

104 
104 
104 

103 

103 
103.5 
103.5 
104 

103.5 
104 










Apr. 26, 2p. m 


.. ..do 








Apr. 26, 4 p. m 

Apr. 27, 9 a. m.... 
Apr. 27, 12 m 


do 

12 c. 0. sub- 
cutaneous. 
do 


0.25 c. c. sub- 
cutaneous. 












Apr. 27, 2 p. m 


do 








Apr. 27, 4 p. m 


do 








Apr, 28, 9 a. m 

Apr. 28, 2p. m 


10 c. c. sub- 
cutaneous. 
do 














Apr. 28, 4 p. m 


do 








Apr. 29, 10 a. m... 






Sheep dead from anthrax in 
3 days. 


Rabbit 32 


Mar. 8, 10 a. m 

Mar. 8, 1 p. m 


0.5 c. c. sub- 
cutaneous. 










104.1 
105.4 
101.8 






Mar. 8, 4.30 p. m.. 










Mar. 9, 9 a. m 





0.2 c. c. sub- 
cutaneous. 





40 



ANTHRAX. 
Table 7. — Experiments with anthraxoin — Continued. 



Animal. 



Rabbit 32. 



Guinea pig . 



Rabbit 33. 



Rabbit 34. 



Rabbit 35. 



Date. 



1908. 
Mar. 10, 9a.m. 
Mar. 11, 9 a. m. 



Mar. 8, 10 a. m.. 
Mar. 9, 10 a. m.. 
Mar. 11, 10 a. m. 

Mar. 16, 9 a. m . . 



Mar. 16, 11 a. m.. 
Mar. 16, 12 m 

Mar. 16,4.30 p.m. 

Mar. 17, 9 a. m 

Mar. 26, 9 a. m. . . . 



Mar. 26, 4p.m. 
Apr. 6, 9 a. m . . 



Apr. 8, 9 a. m. 



Mar. 16, 9 a. m... 



Mar. 16, 11 a. m.. 

Mar, 16, 12 m 

Mar. 16, 1.30 p.m. 
Mar. 17, 9 a. m... 
Mar. 26, 9 a. m... 



Mar. 26, 2 p. m. 
Apr. 6, 9 a. m.. 



Apr, 8, 9 a. m. 



Mar. 16, 9 a. m... 



Mar. 16, 11 a. m. 
Mar. 16, 12 m.... 
Mar. 16, 2 p. m. . 
Mar. 16, 4 p.m.. 
Mar. 17, 9 a. m.. 
Mar. 26, 9 a. m . . 



Mar. 26, 4 p. m. 
Apr. 6, 9 a. m.. . 



Apr. 6, 9 a. m . 



Injections. 



Anthraxoin. 



0.5 c. c. sub- 
cutaneous. 



0.1 c. c. sub- 
cutaneous. 



0.1 c. c. sub- 
cutaneous. 



0.2 c. c. sub- 
cutaneous. 



0.2 e. c. sub- 
cutaneous. 



0.3 c. c. sub- 
cutaneous. 



0.3 c. c. sub- 
cutaneous. 



Anthrax cul- 
ture 



0.2 c. c. sub- 
cutaneous. 



0.01 c. c. sub- 
cutaneous. 



0.01 c. c. sub- 
cutaneous. 



0.01 c. c. sub- 
cutaneous. 



Temper- 
ature. 



105.8 



101 

102.2 
102.5 
104.3 
101.5 
102 

103.5 



102 

103.6 
104.5 
105.3 

102 
102.5 

105 



102 

103.2 

103.5 

104 

105 

102.5 

102 

104.5 



Result and remarks. 



Dead from anthrax in 2 
days. 



Dead from anthrax in 2 
days. 



Animal dead from anthrax 

in 2 days. 



Rabbit stupid. 



Rabbit dead from anthrax 

in 2 days. 



Skin swollen at injection. 

Much swollen from injection. 
Swelling practically gone. 



Dead from anthi-ax in 2 days. 



A COMMERCIAL, VACCINE IN PILL FORM. 

A commercial vaccine which, according to the makers, consists of 
dead anthrax organisms in pill form was also tested. These small 
pills are placed under the skin by means of a trocar and are claimed 
by the makers to produce immunity to anthrax. Microscopic exami- 
nation, as well as cultural and animal experiments, show that the 
claims of the makers, in so far as the vaccine being dead and harm- 
less is concerned, are true. One can easily see with the microscope 
that these little pills consist of dead anthrax bacilli and their spores 



EXPERIMENTS WITH COMMERCIAL. VACCINE. 



41 



held together in pill form by a proper excipient. The writer was 
unable, however, to verify the claim that they produce any im- 
munity, as is shown in Table 2. A rabbit succumbed in six days, but 
as it is very rarely that a rabbit can be immunized by a vaccine, a 
sheep, which animal is easily protected, was also employed, with 
negative results. 

Table 8. — Experiments with commercial vaccine in pill form. 





Date. 


Injections. 

- 


Temper- 
ature. 




Animal. 


Vaccine. 


Anthrax 
culture. 


Result and remarks. 


Rabbit 36 


1908. 
Apr. 7, 9 a. m 

Apr. 8, 9 a. m 

Apr. 9, 4 p. m 


1 pill, sub- 
cutaneous. 

1 pill (dis- 
solved), 
subcuta 
neous, 
do 




°F. 






















do 




0.01 c. c. sub- 
cutaneous. 








Apr. 10, 4 p. m 

Apr. 11, 4 p. m 


1 pill, sub- 
cutaneous. 










104 
104.3 






Am. 13, 4 p. m 










Apr. 15, 4 p. m 






Dead from anthrax in 6 


Sheep 16 


Oct. 17, 9 a. m 

Nov. 2, 9 a. m 

Nov. 4, 9 a. m 


1 pill with 
trocar. 






days. 




0.1 c. c. sub- 
cutaneous. 










Sheep dead from anthrax 












in 48 hours. 



I 



PREPARATION OF AN EPFECTIVE SINGLE VACCINE. 

The single vaccine, like Pasteur vaccine, consists of cultures of 
attenuated anthrax bacilli, the only difference being in the degree 
of attenuation and that, as its name implies, it is applied only once, 
thus requiring a shorter time and only one handling of the animals. 

To prepare such a vaccine or attenuated culture, the virulence of 
an already virulent culture of anthrax was exalted by passage 
through very young animals. A strain can in this way be pro- 
duced which will kill a sucking rabbit in 24 hours. From the heart's 
blood of such a carcass tubes of bouillon are inoculated immediately 
after death. These cultures are then incubated at 42° to 43° C 
for varying periods of time — from 12 to 18 days. On the twelfth 
day a subculture is made from one of the tubes and cultivated at 
35° C. On the thirteenth and succeeding days subcultures are made 
from the remaining tubes until the series has been completed. These 
six subcultures of attenuated bacilli are now tested on guinea pigs 
and rabbits, and on sheep if possible. 



42 ANTHRAX. 

More dependence is to be placed upon the animal test than upon 
the number of days the attenuation process is carried on. A cul- 
ture of proper strength is generally obtained from tubes that have 
been attenuated for about 16 days. The proper culture will be one 
which will not kill rabbits, but which will kill a majority of guinea 
pigs in a delayed period, say 6 or 6 days. Such an animal should 
show no swelling at the point of inoculation, and the bacilli should 
be found only sparingly in the internal organs. When shaken in 
cultures such a vaccine shows a homogeneous clouding of the medium ; 
no flocculi persisting, as occurs in virulent cultures. 

As rabbits and guinea pigs can not be made immune to anthrax 
by vaccination of this kind, these animals are only useful in testing 
the pathogenesis of the cultures. For testing the immunizing prop- 
erty, sheep, which are easily immunized, were employed. Quite a 
number of sheep were used in the experiment to produce a safe, 
efficient single vaccine, and as regards the failures along this line, 
it is only necessary to say that they were entirely due to improper 
attenuation of the cultures. When the proper attenuation was reached 
there was no difficulty in immunizing animals by a single vaccina- 
tion. This was effective against a subsequent, otherwise mortal dose 
of virulent anthrax bacilli, as the following experiments will show. 

TESTS OF THE SINGLE VACCINE. 
Experiment No. 1. 

On April 15, 1909, a visibly pregnant cow was given subcuta- 
neously 1 c. c. of single vaccine. She showed no ill effects whatever 
from the injection. On May 1 the animal was inoculated with 0.2 
c. c. of virulent anthrax bacilli from a 24-hour culture which killed 
a check rabbit and a check cow in 48 hours. On May 19 the cow 
dropped a fully developed calf which when found was dead, lying 
with its head bent under the shoulder. Supposing that the calf had 
been asphyxiated, it and the membranes were buried. However, a 
platinum loop full of the discharge was plated and a pure culture 
of 60,000 anthrax bacilli was obtained. A guinea pig inoculated 
with a culture made from one of these colonies died of anthrax in 
48 hours. The cow remained well, and subsequent daily cultivations 
from vaginal discharges showed no anthrax bacilli. 

This experiment was extremely valuable, not only as showing that 
the cow had been immunized by a single vaccination, but also for 
showing not only that anthrax can be communicated not only to the 
fetus, but that it can be thus communicated by an immune mother. 
It also showed that anthrax bacilli may persist for at least 18 days 
in the body of an immune animal. When the cow was destroyed for 
various reasons on May 25 she was in perfect health, and cultures 



SERUM FOR PRODUCING IMMEDIATE IMMUNITY. 43 

and inoculations made with her blood showed no anthrax bacilli 
present. It is to be regretted that this animal could not have been 
kept for further observations and experiments. 

. Experiment No. 2. 

On September 14, 1909, two sheep were vaccinated with single 
vaccine, each receiving 1 c. c, and showed no sickness therefrom. On 
September 28 each sheep was inoculated subcutaneously with 0.2 c. c. 
of a culture of anthrax bacilli whose virulence had been proven on a 
rabbit in the same experiment. The sheep at no time showed any 
sickness. On November 2 both sheep were again tested with virulent 
bacilli and showed no sickness. 

Experiment No. 3. 

On November 12, 1909, three sheep were vaccinated with 1 c. c. of 
single vaccine and showed no sickness therefrom. On November 27 
one of the animals was tested with 0.2 c. c. of virulent bacilli, and 
on December 1 the other two were similarly tested. They at no 
time showed any sickness. These three sheep, together with the two 
used in experiment No. 2, were used later in experiments to produce 
an antibacterial serum. 

A SERUM FOE, PRODUCING IMMEDIATE IMMUNITY. 

Although the favorable results from vaccination by the Pasteur 
system have been known for a long time, and owing to the cheapness 
of the vaccine it would seem that there is nothing more to be desired, 
the length of time required to produce incmiunity is one drawback in 
its use when one is endeavoring to check an existing outbreak of the 
disease. "^Alien vaccination is practiced a month in advance of the 
animals being turned out to pasture in the spring, the system of 
Pasteur vaccination is the proper one to use. In existing outbreaks, 
however, it is evident that any system that requires a month to be- 
come protectiA^e leaves much to be desired, as many animals could be- 
come infected and die before protection could be afforded them. 

With the end in view of devising a method whereby an immediate 
immunity could be established in existing outbreaks or where an im- 
munity could be brought about in a much shorter time than has been 
possible under the old system of vaccination, the writer has devoted 
a large portion of his time for the past three years. As it had been 
conclusively demonstrated that animals can be immunized by a single 
vaccine, thus cutting down one-half the period necessary for immuni- 
zation by the old system of Pasteur, it was decided to experiment 
upon the production of an antianthrax serum by endeavoring to 



I 



44 ANTHRAX. 

hyperimmunize sheep after the manner of the production of anti- 
hog-cholera serum, with the exception that whereas in the latter work 
virulent blood was used, this was precluded in our experiments owing 
to the dangers attending the handling of large quantities of virulent 
anthrax blood. Instead, the blood used was that drawn from an 
animal immune to anthrax, and not from one sick with anthrax. 

Briefly stated, the writer has produced an antibacterial serum by 
highly immunizing sheep through repeated inoculations, first of at- 
tenuated anthrax bacilli, and following these by inoculations of the 
most virulent races of the bacilli in increasing doses until the animal 
would withstand with impunity fifty thousand times the minimal 
lethal dose. Such a serum will protect a sheep against an otherwise 
mortal dose of bacilli and produce an immediate immunity. It is, 
therefore, a very valuable adjunct in working against the spread of 
the disease in existing outbreaks where the usual vaccination is be- 
ing practiced. The serum will confer a passive immunity immedi- 
ately, and thus protect the animal against fatal infection until the 
vaccine confers an active immunity. And should an animal which 
has been protected by the serum become infected with a virulent 
anthrax bacillus, the results of this infection will be the production 
of a much stronger immunity than the vaccine and serum would 
otherwise confer. 

It is evident that if a single vaccine, or even a double vaccine, can 
be used in conjunction with an antibacterial serum to produce im- 
mediate passive immunity which will persist until the vaccine has 
had time to bring about active immunity a long step will have been 
made in controlling this formidable disease. The experiments with 
this end in view were carried out upon sheep as follows: 

EXPERIMENTS WITH THE SERUM. 
Experiment No. 1. 

On December 15, 1909, a sheep immunized with single vaccine and 
a subsequent inoculation with virulent bacilli (see Experiments Nos. 
2 and 3 with single vaccine) was bled from the carotid artery — the 
femoral artery being small and deep-seated — ^by means of a glass 
cannula and rubber tubing. Wlien the blood had clotted, 500 c. c. of 
serum were decanted and injected into the inguinal regions of another 
sheep which had been similarly immunized. Microscopic examina- 
tions and inoculations into guinea pigs of this blood showed it to be 
free of anthrax bacilli. The injected sheep was lame in both hind 
legs the next day, but this lameness disappeared when the complete 
absorption of the injected blood had taken place, and no abscess 
formed. On January 12 this sheep was bled from the carotid artery. 



EXPERIMENTS WITH THE SERUM. 45 

and when the serum was collected it was preserved by adding to it 
one part of a 5 per cent solution of carbolic acid to each nine parts 
of serum. In making this addition of carbolic acid some coagulation 
will occur if the preservative and serum be not poured simultaneously 
into another vessel and stirred by a helper. 

The above serum was without protective properties. All animals 
upon which it was used died of anthrax, as follows : 

On January 13, 1910, 12 guinea pigs received increasing doses of 
serum from 0.1 up to 3 c. c. and simultaneously 0.1 c. c. of virulent 
anthrax culture. All of these animals died of anthrax in 48 hours. 

On January IT, 1910, the amounts of serum were increased to 4 c. c, 
5 c. c, 6 c. c, 7 c. c, 8 c. c, 9 c. c, and 10 c. c, with a simultaneous dose 
of 0.1 c. c. of virulent culture. The 7 animals thus tested also died in 
48 hours. 

On February 5, 1910, a sheep received subcutaneously 24 c. c. of 
the serum and 0.2 c. c. of virulent culture. A control rabbit died in 
48 hours and the sheep died of anthrax in three days. 

On February 9, 10, and 11 another sheep received daily 12 c. c. of 
the serum and on the 12th a test dose of virulent bacilli. This sheep 
lived four days and then died of anthrax. 

These experiments demonstrated that the modified hog-cholera 
method of serum production could not be applied to anthrax, and 
it was then decided to try and bring about hyperimmunization by 
increasing doses of virulent bacilli repeated over a long period. The 
two sheep used in experiment No. 2 with single vaccine were em- 
ployed for this purpose as follows : 

Experiment No. 2. 

On September 14, 1909, two sheep were vaccinated with 1 c. c. of 
single vaccine. On September 28 they were given a test dose of 0.2 
c. c. of virulent bacilli, and remained well, while a check rabbit died 
in 48 hours. On November 2 each sheep again received 0.2 c. c. of 
virulent bacilli. On March 4, 1910, each sheep received 0.3 c. c. of 
virulent bacilli. On March 18 they received 1 c. c. of virulent bacilli, 
and on April 2 each sheep received 5 c. c. of virulent bacilli. A 
period of about seven months was thus consumed. 

On April 14, 1910, these sheep were bled from the carotid artery, 
producing 1,900 c. c. of serum, to which was added one part of a 5 
per cent solution of carbolic acid to each nine parts of serum as a 
preservative. Prior to the addition of the preservative tests for the 
presence of anthrax bacilli were made upon guinea pigs and by plate 
cultures. None were found. 

This serum was used upon sheep in connection with virulent an- 
thrax bacilli as shown in Table 9. 



46 



ANTHRAX. 
Table 9. — Experiments tvitU antianthrax serum. 



Animal. 



Date. 



Injections. 



Immune serum. 



Virulent culture. 



Results and remarks. 



Sheep 17 

Rabbit (control) 
Sheep 18 



1910. 
Apr. 29 
May 16 
..do 



5 CO. subcutaneous.. 



Rabbit (control) 
Sheep 19 



Rabbit(control) 
Sheep 20 



Sheep 21 . 



Rabbit (control) 
Sheep 22 



Sheep 23. 
Sheep 24. 



Sheep 25 (con- 
trol). 



Rabbit (control) 



May 21 
June 20 
July 6 
May 21 

May 24 
June 20 
July 6 
May 24 

May 27 
June 20 
July 6 

May 27 
June 20 
July 6 
June 20 

June 1 
June 18 
July 6 

June 1 
June IS 
July 6 

June 1 
June 18 
July 6 

June 18 



July G 



10 c. c. subcutaneous. 

do 

do 



10 c. c. subcutaneous. 

....do 

...Ao 



10 c. c. subcutaneous. 
do , 



10c. c. subcutaneous. 
do 



5 CO. subcutaneous. 
10 c. c. subcutaneous. 
do 

5 c. c. subcutaneous . . 
10 c. c. subcutaneous. 
do 

5 c. c. subcutaneous. 
10 c. c. subcutaneous 
do 



0. 2 c. c. subcutaneous. . . 
. 2 c. c. subcutaneous. . . 
. 1 c. c. subcutaneous. . . 

. 1 c. c. subcutaneous. . . 
. 2 c. c. subcutaneous. . . 
. 5 c. c. subcutaneous . . . 
. 1 c. c. virulent culture . 

. 1 c. c. subcutaneous . . - 
. 1 c. c. subcutaneous . . . 
. 5 c. c. subcutaneous . . . 
• Ice. virulent culture. 

. 5 c. c. single vaccine. . . 
. 1 c. c. virulent culture. 
. 5 c. c. virulent culture. 

. 5 0. c. single vaccine. . . 
. 1 c. c. virulent culture. 
.5 c. c. virulent culture. 
.Ice. virulent culture. 

1. c. c. single vaccine. . . 
. 1 c. c. virulent culture. 
. 5 c. c. virulent culture. 

1 . c. c. single vaccine. . . 
. 5 c. c. virulent culture. 
. 5 c. c. virulent culture. 

1. c. c. single vaccine. . . 
. 1 c. c. virulent culture. 
. 5 c. c. virulent culture. 

. 1 c. c. virulent culture. 



. 1 c. c. virulent culture 



Sheep remains well. 
Rabbit dies in 48 hours. 

Not sick. 
No bad result. 
Remained well. 
Died in 48 hours. 



Remained well. 
Died in 48 hours. 

Remained well. 

Do. 
Dead in 48 hours. 

Remained well. 



Do. 



Do. 



Died of anthrax in 48 hours 
(control for sheep 22, 23, 
and 24, for inoculations 
June 18). 

Died of anthrax in 48 hours 
(control for sheep 22, 23, 
and 24, for inoculations on 
July 6. 



No doubt the period of time occupied in hyperimmunizing the 
sheep which produced this highly protective serum can be very much 
lessened — probably one-half. It will be noted that the time covered 
was about seven months. This was not intentional, but as the sheep 
had already been immunized for another object, and not used, it was 
decided to carry out the idea of greatly increasing their immunity 
by the method followed. It will be seen by referring to the table 
that an idle period of about three months elapsed between November, 
1909, and February, 1910, which might just as well have been em- 
ployed for the purpose. It is also possible that by giving larger 
doses of virulent bacilli the immunizing property of the serum may 
be considerably increased. 

That the degree of immunity can be established by the number of 
inoculations and the quantity of virulent culture used was clearly 
shown in sheep Nos. 18, 19, 20, 21, 22, 23, and 24. They had received 



coNCLusioisr. 47 

three injections of serum and three inoculations with bacilli, in very 
small doses, and while they themselves were immune (see Table 9) 
their blood, when tested after slaughter on July 18, did not protect 
other sheep. 

It is also highly probable that sheep will produce a more effective 
antianthrax serum for their own species, and cattle for cattle, horses 
for horses, etc. ; that is, the sera should be homologous. However, no 
effort was made to test this principle, as the great expense attending 
such experiments precluded their being carried out. 

CONCLUSION. 

The writer does not advise the abandonment of the old Pasteur 
system of vaccination against anthrax when it is practiced upon ani- 
mals before they are turned out on the pastures in the spring of the 
year. When animals are dying, however, vaccination alone requires 
too long a period to protect, and it is in these outbreaks that the anti- 
anthrax serum should be used in conjunction with vaccine. The ex- 
periments have shown that a single vaccine may be used with good 
results. Where it is desired, however, the serum may be used simul- 
taneously with the double vaccine or with a single vaccine. 



^ 



